Nucleic acids encoding hematopoietic stem cells receptors flk-1

ABSTRACT

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. 1(flk-2) and FIG. 2 (flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. 1(flk-2) and FIG. 2 (flk-1); ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

The invention described in this application was made with U.S. government support from Grant Numbers R01-CA45339 and R01-DK42989 awarded by the National Institutes of Health. The government has certain rights in this invention.

This is a continuation of copending application(s) Ser. No. 07/813,593 filed on Dec. 24, 1991, issued as U.S. Pat No. 5,185,438, which is a continuation-in-part of Ser. No. 793,065 filed Nov. 15, 1991, which is a continuation-in-part of Ser. No. 728,913 filed Jun. 28, 1991 now abandoned, which is a continuation-in-part of Ser. No. 679,666 filed Apr. 2, 1991 now abandoned, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to hematopoietic stem cell receptors, ligands for such receptors, and nucleic acid molecules encoding such receptors and ligands.

BACKGROUND OF THE INVENTION

The mammalian hematopoietic system comprises red and white blood cells. These cells are the mature cells that result from more primitive lineage-restricted cells. The cells of the hematopoietic system have been reviewed by Dexter and Spooncer in the Annual Review of Cell Biology 3, 423-441 (1987).

The red blood cells, or erythrocytes, result from primitive cells referred to by Dexter and Spooncer as erythroid burst-forming units (BFU-E). The immediate progeny of the erythroid burst-forming units are called erythroid colony-forming units (CFU-E).

The white blood cells contain the mature cells of the lymphoid and myeloid systems. The lymphoid cells include B lymphocytes and T lymphocytes. The B and T lymphocytes result from earlier progenitor cells referred to by Dexter and Spooncer as preT and preB cells.

The myeloid system comprises a number of cells including granulocytes, platelets, monocytes, macrophages, and megakaryocytes. The granulocytes are further divided into neutrophils, eosinophils, basophils and mast cells.

Each of the mature hematopoietic cells are specialized for specific functions. For example, erythrocytes are responsible for oxygen and carbon dioxide transport. T and B lymphocytes are responsible for cell- and antibody-mediated immune responses, respectively. Platelets are involved in blood clotting. Granulocytes and macrophages act generally as scavengers and accessory cells in the immune response against invading organisms and their by-products.

At the center of the hematopoietic system lie one or more totipotent hematopoietic stem cells, which undergo a series of differentiation steps leading to increasingly lineage-restricted progenitor cells. The more mature progenitor cells are restricted to producing one or two lineages. Some examples of lineage-restricted progenitor cells mentioned by Dexter and Spooncer include granulocyte/macrophage colony-forming cells (GM-CFC), megakaryocyte colony-forming cells (Meg-CFC), eosinophil colony-forming cells (Eos-CFC), and basophil colony-forming cells (Bas-CFC). Other examples of progenitor cells are discussed above.

The hematopoietic system functions by means of a precisely controlled production of the various mature lineages. The totipotent stem cell possesses the ability both to self renew and to differentiate into committed progenitors for all hematopoietic lineages. These most primitive of hematopoietic cells are both necessary and sufficient for the complete and permanent hematopoietic reconstitution of a radiation-ablated hematopoietic system in mammals. The ability of stem cells to reconstitute the entire hematopoietic system is the basis of bone marrow transplant therapy.

It is known that growth factors play an important role in the development and operation of the mammalian hematopoietic system. The role of growth factors is complex, however, and not well understood at the present time. One reason for the uncertainty is that much of what is known about hematopoietic growth factors results from in vitro experiments. Such experiments do not necessarily reflect in vivo realities.

In addition, in vitro hematopoiesis can be established in the absence of added growth factors, provided that marrow stromal cells are added to the medium. The relationship between stromal cells and hematopoietic growth factors in vivo is not understood. Nevertheless, hematopoietic growth factors have been shown to be highly active in vivo.

From what is known about them, hematopoietic growth factors appear to exhibit a spectrum of activities. At one end of the spectrum are growth factors such as erythropoietin, which is believed to promote proliferation only of mature erythroid progenitor cells. In the middle of the spectrum are growth factors such as IL-3, which is believed to facilitate the growth and development of early stem cells as well as of numerous progenitor cells. Some examples of progenitor cells induced by IL-3 include those restricted to the granulocyte/macrophage, eosinophil, megakaryocyte, erythroid and mast cell lineages.

At the other end of the spectrum is the hematopoietic growth factor that, along with the corresponding receptor, was discussed in a series of articles in the Oct. 5, 1990 edition of Cell. The receptor is the product of the W locus, c-kit, which is a member of the class of receptor protein tyrosine kinases. The ligand for c-kit, which is referred to by various names such as stem cell factor (SCF) and mast cell growth factor (MGF), is believed to be essential for the development of early hematopoietic stem cells and cells restricted to the erythroid and mast cell lineages in mice; see, for example, Copeland et al., Cell 63, 175-183 (1990).

It appears, therefore, that there are growth factors that exclusively affect mature cells. There also appear to be growth factors that affect both mature cells and stem cells. The growth factors that affect both types of cells may affect a small number or a large number of mature cells.

There further appears to be an inverse relationship between the ability of a growth factor to affect mature cells and the ability of the growth factor to affect stem cells. For example, the c-kit ligand, which stimulates a small number of mature cells, is believed to be more important in the renewal and development of stem cells then is IL-3, which is reported to stimulate proliferation of many mature cells (see above).

Prior to the present specification, there have been no reports of growth factors that exclusively stimulate stem cells in the absence of an effect on mature cells. The discovery of such growth factors would be of particular significance.

As mentioned above, c-kit is a protein tyrosine kinase (pTK). It is becoming increasingly apparent that the protein tyrosine kinases play an important role as cellular receptors for hematopoietic growth factors. Other receptor pTKs include the receptors of colony stimulating factor 1(CSF-1) and PDGF.

The pTK family can be recognized by the presence of several conserved amino acid regions in the catalytic domain. These conserved regions are summarized by Hanks et al. in Science 241, 42-52 (1988), see FIG. 1 starting on page 46 and by Wilks in Proc. Natl. Acad. Sci. USA 86, 1603-1607 (1989), see FIG. 2 on page 1605.

Additional protein tyrosine kinases that represent hematopoietic growth factor receptors are needed in order more effectively to stimulate the self-renewal of the totipotent hematopoietic stem cell and to stimulate the development of all cells of the hematopoietic system both in vitro and in vivo. Novel hematopoietic growth factor receptors that are present only on primitive stem cells, but are not present on mature progenitor cells, are particularly desired. Ligands for the novel receptors are also desirable to act as hematopoietic growth factors. Nucleic acid sequences encoding the receptors and ligands are needed to produce recombinant receptors and ligands.

SUMMARY OF THE INVENTION

These and other objectives as will be apparent to those with ordinary skill in the art have been met by providing isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. 1 (flk-2, SEQIDNO:1 and 2) and FIG. 2 (flk-1, SEQIDNO:3 and 4); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. 1 (flk-2) and FIG. 2 (flk-1); ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

DESCRIPTION OF THE FIGURES

FIG. 1a through FIG. 1E shows the cDNA and amino acid sequences of flk-2(SEQ ID NO:1 and 2). The amino acid residues occur directly below the nucleotides in the open reading frame. Amino acids 1-27 constitute the hydrophobic leader sequence. Amino acids 28-544 constitute the extracellular receptor domain. Amino acids 545-564 constitute the transmembrane region. The remainder of the amino acids constitute the intracellular catalytic domain. The following amino acid residues in the intracellular domain are catalytic sub-domains identified by Hanks (see above): 545-564, 618-623, 811-819, 832-834, 857-862, 872-878. The sequence at residues 709-785 is a signature sequence characteristic of flk-2. The protein tyrosine kinases generally have a signature sequence in this region. For the purposes of this specification and the claims, it is understood that all references to FIG. 1 includes FIGS. 1a through 1e.

FIG. 2a through FIGS. 2g shows the cDNA and amino acid sequences of flk-1 (SEQ ID NO:3 and 4). Amino acid residue 763-784 constitute the transmembrane region of flk-1. For the purposes of this specification and the claims, it is understood that all references to FIG. 2 includes FIGS. 2a through 2g.

FIG. 3 shows the time response of binding between stromal cells (2018) and APtag-flk-2 as well as APtag-flk-1. APtag without receptor (SEAP) is used as a control. See Example 8.

FIG. 4 shows the dose response of binding between stromal cells (2018) and APtag-flk-2 as well as APtag-flk-1. APtag without receptor (SEAP) is used as a control. See Example 8.

DETAILED DESCRIPTION OF THE INVENTION Receptors

In one embodiment, the invention relates to an isolated mammalian nucleic acid molecule encoding a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

The nucleic acid molecule may be a DNA, cDNA, or RNA molecule. The mammal in which the nucleic acid molecule exists may be any mammal, such as a mouse, rat, rabbit, or human.

The nucleic acid molecule encodes a protein tyrosine kinase (pTK). Members of the pTK family can be recognized by the conserved amino acid regions in the catalytic domains. Examples of pTK consensus sequences have been provided by Hanks et al. in Science 241, 42-52 (1988); see especially FIG. 1 starting on page 46 and by Wilks in Proc. Natl. Acad. Sci. USA 86, 1603-1607 (1989); see especially FIG. 2 on page 1605. A methionine residue at position 205 in the conserved sequence WMAPES is characteristic of pTK's that are receptors.

The Hanks et al article identifies eleven catalytic sub-domains containing pTK consensus residues and sequences. The pTKs of the present invention will have most or all of these consensus residues and sequences.

Some particularly strongly conserved residues and sequences are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         Conserved Residues and Sequences in pTKs.sup.1                                               Residue or                                                                               Catalytic                                              Position.sup.2                                                                               Sequence  Domain                                                 ______________________________________                                          50           G         I                                                       52           G         I                                                       57           V         I                                                       70           A         II                                                      72           K         II                                                      91           E         III                                                    166           D         VI                                                     171           N         VI                                                     184-186       DFG       VII                                                    208           E         VIII                                                   220           D         IX                                                     225           G         IX                                                     280           R         XI                                                     ______________________________________                                          1. See Hanks et al., Science 241, 42-52 (1988)                                 2. Adjusted in accordance with Hanks et al., Id.                         

A pTK of the invention may contain all thirteen of these highly conserved residues and sequences. As a result of natural or synthetic mutations, the pTKs of the invention may contain fewer than all thirteen strongly conserved residues and sequences, such as 11, 9, or 7 such sequences.

The receptors of the invention generally belong to the same class of pTK sequences that c-kit belongs to. It has surprisingly been discovered, however, that a new functional class of receptor pTKs exists. The new functional class of receptor pTKs is expressed in primitive hematopoietic cells, but not expressed in mature hematopoietic cells.

For the purpose of this specification, a primitive hematopoietic cell is totipotent, i.e. capable of reconstituting all hematopoietic blood cells in vivo. A mature hematopoietic cell is non-self-renewing, and has limited proliferative capacity--i.e., a limited ability to give rise to multiple lineages. Mature hematopoietic cells, for the purposes of this specification, are generally capable of giving rise to only one or two lineages in vitro or in vivo.

It should be understood that the hematopoietic system is complex, and contains many intermediate cells between the primitive totipotent hematopoietic stem cell and the totally committed mature hematopoietic cells defined above. As the stem cell develops into increasingly mature, lineage-restricted cells, it gradually loses its capacity for self-renewal.

The receptors of the present invention may and may not be expressed in these intermediate cells. The necessary and sufficient condition that defines members of the new class of receptors is that they are present in the primitive, totipotent stem cell or cells, and not in mature cells restricted only to one or, at most, two lineages.

An example of a member of the new class of receptor pTKs is called fetal liver kinase 2 (flk-2) after the organ in which it was found. There is approximately 1totipotent stem cell per 10⁴ cells in mid-gestation (day 14) fetal liver in mice. In addition to fetal liver, flk-2 is also expressed in fetal spleen, fetal thymus, adult brain, and adult marrow.

For example, flk-2 is expressed in individual multipotential CFU-Blast colonies capable of generating numerous multilineage colonies upon replating. It is likely, therefore, that flk-2 is expressed in the entire primitive (i.e. self-renewing) portion of the hematopoietic hierarchy. This discovery is consistent with flk-2 being important in transducing putative self-renewal signals from the environment.

It is particularly relevant that the expression of flk-2 mRNA occurs in the most primitive thymocyte subset. Even in two closely linked immature subsets that differ in expression of the IL-2 receptor, flk-2 expression segregates to the more primitive subset lacking an IL-2 receptor. The earliest thymocyte subset is believed to be uncommitted. Therefore, the thymocytes expressing flk-2 may be multipotential. flk-2 is the first receptor tyrosine kinase known to be expressed in the T-lymphoid lineage.

The fetal liver mRNA migrates relative to 285 and 185 ribosomal bands on formaldehyde agarose gels at approximately 3.5 kb while the brain message is considerably larger. In adult tissues, flk-2 m-RNA from both brain and bone marrow migrated at approximately 3.5 kb.

A second pTK receptor is also included in the present invention. This second receptor, which is called fetal liver kinase 1(flk-1), is not a member of the same class of receptors as flk-2, since flk-1 may be found in some more mature hematopoietic cells. The amino acid sequence of flk-1is given in FIG. 2 (SEQ ID NO:4).

The present invention includes the flk-1 receptor as well as DNA, cDNA and RNA encoding flk-1. The DNA sequence of flk-1is also given in FIG. 2. Flk-1may be found in the same organs as flk-2, as well as in fetal brain, stomach, kidney, lung, heart and intestine; and in adult kidney, heart, spleen, lung, muscle, and lymph nodes.

The receptor protein tyrosine kinases of the invention are known to be divided into easily found domains. The DNA sequence corresponding to the pTKs encode, starting at their 5'-ends, a hydrophobic leader sequence followed by a hydrophilic extracellular domain, which binds to, and is activated by, a specific ligand. Immediately downstream from the extracellular receptor domain, is a hydrophobic transmembrane region. The transmembrane region is immediately followed by a basic catalytic domain, which may easily be identified by reference to the Hanks et al. and Wilks articles discussed above.

The present invention includes the extracellular receptor domain lacking the transmembrane region and catalytic domain. Preferably, the hydrophobic leader sequence is also removed from the extracellular domain. In the case of flk-2, the hydrophobic leader sequence includes amino acids 127.

These regions and domains may easily be visually identified by those having ordinary skill in the art by reviewing the amino acid sequence in a suspected pTK and comparing it to known pTKs. For example, referring to FIG. 1, the transmembrane region of flk-2, which separates the extracellular receptor domain from the catalytic domain, is encoded by nucleotides 1663 (T) to 1722 (C). These nucleotides correspond to amino acid residues 545 (Phe) to 564 (Cys). The amino acid sequence between the transmembrane region and the catalytic sub-domain (amino acids 618-623) identified by Hanks et al. as sub-domain I (i.e., GXGXXG) is characteristic of receptor protein tyrosine kinases.

The extracellular domain may also be identified through commonly recognized criteria of extracellular amino acid sequences. The determination of appropriate criteria is known to those skilled in the art, and has been described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. USA 78, 3824-3828 (1981); Kyte et al, J. Mol. Biol. 157, 105-132 (1982); Emini, J. Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed characteristic of extracellular domains.

As will be discussed in more detail below, the nucleic acid molecules that encode the receptors of the invention may be inserted into known vectors for use in standard recombinant DNA techniques. Standard recombinant DNA techniques are those such as are described in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and by Ausubel et al., Eds, "Current Protocols in Molecular Biology," Green Publishing Associates and Wiley-Interscience, New York (1987). The vectors may be circular (i.e. plasmids) or non-circular. Standard vectors are available for cloning and expression in a host. The host may be prokaryotic or eucaryotic. Prokaryotic hosts are preferably E. coli. Preferred eucaryotic hosts include yeast, insect and mammalian cells. Preferred mammalian cells include, for example, CHO, COS and human cells.

Ligands

The invention also includes ligands that bind to the receptor pTKs of the invention. In addition to binding, the ligands stimulate the proliferation of additional primitive stem cells, differentiation into more mature progenitor cells, or both.

The ligand may be a growth factor that occurs naturally in a mammal, preferably the same mammal that produces the corresponding receptor. The growth factor may be isolated and purified, or be present on the surface of an isolated population of cells, such as stromal ells.

The ligand may also be a molecule that does not occur naturally in a mammal. For example, antibodies, preferably monoclonal, raised against the receptors of the invention or against anti-ligand antibodies mimic the shape of, and act as, ligands if they constitute the negative image of the receptor or anti-ligand antibody binding site. The ligand may also be a non-protein molecule that acts as a ligand when it binds to, or otherwise comes into contact with, the receptor.

In another embodiment, nucleic acid molecules encoding the ligands of the invention are provided. The nucleic acid molecule may be RNA, DNA or cDNA.

Stimulating Proliferation of Stem Cells

The invention also includes a method of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells as defined above. The method comprises contacting the stem cells With a ligand in accordance with the present invention. The stimulation of proliferation and/or differentiation may occur in vitro or in vivo.

The ability of a ligand according to the invention to stimulate proliferation of stem cells in vitro and in vivo has important therapeutic applications. Such applications include treating mammals, including humans, whose primitive stem cells do not sufficiently undergo self-renewal. Example of such medical problems include those that occur when defects in hematopoietic stem cells or their related growth factors depress the number of white blood cells. Examples of such medical problems include anemia, such as macrocytic and aplastic anemia. Bone marrow damage resulting from cancer chemotherapy and radiation is another example of a medical problem that would be helped by the stem cell factors of the invention.

Functional Equivalents

The invention includes functional equivalents of the pTK receptors, receptor domains, and ligands described above as well as of the nucleic acid sequences encoding them. A protein is considered a functional equivalent of another protein for a specific function if the equivalett protein is immunologically cross-reactive with, and has the same function as, the receptors and ligands of the invention. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.

For example, it is possible to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids known normally to be equivalent are:

(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);

(b) Asn(N) Asp(D) Glu(E) Gln(Q);

(c) His(H) Arg(R) Lys(K);

(d) Met(M) Leu(L) Ile(I) Val(V); and

(e) Phe(F) Tyr(Y) Trp(W).

Substitutions, additions and/or deletions in the receptors and ligands may be made as long as the resulting equivalent receptors and ligands are immunologically cross reactive with, and have the same function as, the native receptors and ligands.

The equivalent receptors and ligands will normally have substantially the same amino acid sequence as the native receptors and ligands. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions and/or deletions is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in the amino acid sequence of the native receptors and ligands are substituted for, added to, or deleted from.

Equivalent nucleic acid molecules include nucleic acid sequences that encode equivalent receptors and ligands as defined above. Equivalent nucleic acid molecules also include nucleic acid sequences that differ from native nucleic acid sequences in ways that do not affect the corresponding amino acid sequences.

ISOLATION OF NUCLEIC ACID MOLECULES AND PROTEINS Isolation of Nucleic Acid Molecules Encoding Receptors

In order to produce nucleic acid molecules encoding mammalian stem cell receptors, a source of stem cells is provided. Suitable sources include fetal liver, spleen, or thymus cells or adult marrow or brain cells.

For example, suitable mouse fetal liver cells may be obtained at day 14 of gestation. Mouse fetal thymus cells may be obtained at day 14-18, preferably day 15, of gestation. Suitable fetal cells of other mammals are obtained at gestation times corresponding to those of mouse.

Total RNA is prepared by standard procedures from stem cell receptor-containing tissue. The total RNA is used to direct cDNA synthesis. Standard methods for isolating RNA and synthesizing cDNA are provided in standard manuals of molecular biology such as, for example, in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and in Ausubel et al., (Eds), "Current Protocols in Molecular Biology," Greene Associates/Wiley Interscience, New York (1990).

The cDNA of the receptors is amplified by known methods. For example, the cDNA may be used as a template for amplification by polymerase chain reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or Mullis et al., U.S. Pat. No. 4,683,195. The sequences of the oligonucleotide primers for the PCR amplification are derived from the sequences of known receptors, such as from the sequences given in FIGS. 1 and 2 for flk-2 and flk-1, respectively, preferably from flk-2. The oligonucleotides are synthesized by methods known in the art. Suitable methods include those described by Caruthers in Science 230, 281-285 (1985).

In order to isolate the entire protein-coding regions for the receptors of the invention, the upstream oligonucleotide is complementary to the sequence at the 5, end, preferably encompassing the ATG start codon and at least 5-10 nucleotides upstream of the start codon. The downstream oligonucleotide is complementary to the sequence at the 3' end, optionally encompassing the stop codon. A mixture of upstream and downstream oligonucleotides are used in the PCR amplification. The conditions are optimized for each particular primer pair according to standard procedures. The PCR product is analyzed by electrophoresis for the correct size cDNA corresponding to the sequence between the primers.

Alternatively, the coding region may be amplified in two or more overlapping fragments. The overlapping fragments are designed to include a restriction site permitting the assembly of the intact cDNA from the fragments.

The amplified DNA encoding the receptors of the invention may be replicated in a wide variety of cloning vectors in a wide variety of host cells. The host cell may be prokaryotic or eukaryotic. The DNA may be obtained from natural sources and, optionally, modified, or may be synthesized in whole or in part.

The vector into which the DNA is spliced may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable prokaryotic cloning vectors include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages.

Isolation of Receptors

DNA encoding the receptors of the invention are inserted into a suitable vector and expressed in a suitable prokaryotic or eucaryotic host. Vectors for expressing proteins in bacteria, especially E. coli, are known. Such vectors include the PATH vectors described by Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-galactosidase (pEX); lambda Pr; maltose binding protein (pMAL); and glutathione S-transferase (pGST) - see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).

Vectors useful in yeast are available. A suitable example is the 2u plasmid.

Suitable vectors for use in mammalian cells are also known. Such vectors include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and vectors derived from combination of plasmids and phage DNA.

Further eukaryotic expression vectors are known in the art (e.g., P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann and P. A. Sharp, "Amplification And Expression Of Sequences Cotransfected with A Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S. I. Scahill et al, "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Natl. Acad. Sci. USA 80. 4654-4659 (1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77, 4216-4220, (1980).

The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

Vectors containing the receptor-encoding DNA and control signals are inserted into a host cell for expression of the receptor. Some useful expression host cells include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces. Suitable eukaryotic cells include yeast and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.

The human homologs of the mouse receptors described above are isolated by a similar strategy. RNA encoding the receptors are obtained from a source of human cells enriched for primitive stem cells. Suitable human cells include fetal spleen, thymus and liver cells, and umbilical cord blood as well as adult brain and bone marrow cells. The human fetal cells are preferably obtained on the day of gestation corresponding to mid-gestation in mice. The amino acid sequences of the human flk receptors as well as of the nucleic acid sequences encoding them are homologous to the amino acid and nucleotide sequences of the mouse receptors.

In the present specification, the sequence of a first protein, such as a receptor or a ligand, or of a nucleic acid molecule that encodes the protein, is considered homologous to a second protein or nucleic acid molecule if the amino acid or nucleotide sequence of the first protein or nucleic acid molecule is at least about 30% homologous, preferably at least about 50% homologous, and more preferably at least about 65% homologous to the respective sequences of the second protein or nucleic acid molecule. In the case of proteins having high homology, the amino acid or nucleotide sequence of the first protein or nucleic acid molecule is at least about 75% homologous, preferably at least about 85% homologous, and more preferably at least about 95% homologous to the amino acid or nucleotide sequence of the second protein or nucleic acid molecule.

Combinations of mouse oligonucleotide pairs are used as PCR primers to amplify the human homologs from the cells to account for sequence divergence. The remainder of the procedure for obtaining the human flk homologs are similar to those described above for obtaining mouse flk receptors. The less than perfect homology between the human flk homologs and the mouse oligonucleotides is taken into account in determining the stringency of the hybridization conditions.

Assay for expression of Receptors on Stem Cells

In order to demonstrate the expression of flk receptors on the surface of primitive hematopoietic stem cells, antibodies that recognize the receptor are raised. The receptor may be the entire protein as it exists in nature, or an antigenic fragment of the whole protein. Preferably, the fragment comprises the predicted extracellular portion of the molecule.

Antigenic fragments may be identified by methods known in the art. Fragments containing antigenic sequences may be selected on the basis of generally accepted criteria of potential antigenicity and/or exposure. Such criteria include the hydrophilicity and relative antigenic index, as determined by surface exposure analysis of proteins. The determination of appropriate criteria is known to those skilled in the art, and has been described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. USA 78, 3824-3828 (1981); Kyte et al, J. Mol. Biol. 157. 105-132 (1982); Emini, J. Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed are selected preferentially over domains predicted to be more hydrophobic or hidden.

The proteins and fragments of the receptors to be used as antigens may be prepared by methods known in the art. Such methods include isolating or synthesizing DNA encoding the proteins and fragments, and using the DNA to produce recombinant proteins, as described above.

Fragments of proteins and DNA encoding the fragments may be chemically synthesized by methods known in the art from individual amino acids and nucleotides. Suitable methods for synthesizing protein fragments are described by Stuart and Young in "Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984). Suitable methods for synthesizing NA fragments are described by Caruthers in Science 230, 281-285 (1985).

If the receptor fragment defines the epitope, but is too short to be antigenic, it may be conjugated to a carrier molecule in order to produce antibodies. Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.

The antibodies are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256, 495-497 (1975) and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al in Science 246, 1275-1281 (1989).

Polyclonal or monoclonal antisera shown to be reactive with receptor-encoded native proteins, such as with flk-1 and flk-2 encoded proteins, expressed on the surface of viable cells are used to isolate antibody-positive cells. One method for isolating such cells is flow cytometry; see, for example, Loken et al., European patent application 317,156. The cells obtained are assayed for stem cells by engraftment into radiation-ablated hosts by methods known in the art; see, for example, Jordan et al., Cell 61, 953-963 (1990).

Criteria for Novel stem Cell Receptor Tyrosine Kinases Expressed in Stem Cells

Additional novel receptor tyrosine kinase cDNAs are obtained by amplifying cDNAs from stem cell populations using oligonucleotides as PCR primers; see above. Examples of suitable oligonucleotides are PTK1 and PTK2, which were described by Wilks et al. in Proc. Natl. Acad. Sci. USA 86, 1603-1607 (1989). Novel cDNA is selected on the basis of differential hybridization screening with probes representing known kinases. The cDNA clones hybridizing only at low stringency are selected and sequenced. The presence of the amino acid triplet DFG confirms that the sequence represents a kinase. The diagnostic methionine residue in the WMAPES motif is indicative of a receptor-like kinase, as described above. Potentially novel sequences obtained are compared to available sequences using databases such as Genbank in order to confirm uniqueness. Gene-specific oligonucleotides are prepared as described above based on the sequence obtained. The oligonucleotides are used to analyze stem cell enriched and depleted populations for expression. Such cell populations in mice are described, for example, by Jordan et al. in Cell 61, 953-956 (1990); Ikuta et al. in Cell 62, 863-864 (1990); Spangrude et al. in Science 241, 58-62 (1988); and Szilvassy et al. in Blood 74, 930-939 (1989). Examples of such human cell populations are described as CD33⁻ CD34⁺ by Andrews et al. in the Journal of Experimental Medicine 169, 1721-1731 (1989). Other human stem cell populations are described, for example, in Civin et al., European Patent Application 395,355 and in Loken et al., European Patent Application 317,156.

lsolating Ligands and Nucleic Acid Molecules Encoding Ligands

Cells that may be used for obtaining ligands include stromal cells, for example stromal cells from fetal liver, fetal spleen, fetal thymus and fetal or adult bone marrow. Cell lines expressing ligands are established and screened.

For example, cells such as stromal (non-hematopoietic) cells from fetal liver are immortalized by known methods. Examples of known methods of immortalizing cells include transduction with a temperature sensitive SV40 T-antigen expressed in a retroviral vector. Infection of fetal liver cells with this virus permits the rapid and efficient establishment of multiple independent cell lines. These lines are screened for ligand activity by methods known in the art, such as those outlined below.

Ligands for the receptors of the invention, such as flk-1 and flk-2, may be obtained from the cells in several ways. For example, a bioassay system for ligand activity employs chimeric tagged receptors; see, for example, Flanagan et al., Cell 63, 185-194 (1990). One strategy measures ligand binding directly via a histochemical assay. Fusion proteins comprising the extracellular receptor domains and secretable alkaline phosphatase (SEAP) are constructed and transfected into suitable cells such as NIH/3T3 or COS cells. Flanagan et al. refer to such DNA or amino acid constructs as APtag followed by the name of the receptor--i.e. APtag-c-kit. The fusion proteins bind with high affinity to cells expressing surface-bound ligand. Binding is detectable by the enzymatic activity of the alkaline phosphatase secreted into the medium. The bound cells, which are often stromal cells, are isolated from the APtag-receptor complex.

For example, some stromal cells that bind APtag-flk1 and APtag-flk2 fusion proteins include mouse fetal liver cells (see example 1); human fetal spleen cells (see example 3); and human fetal liver (example 3). Some stromal fetal thymus cells contain flk-1 ligand (example 3).

To clone the cDNA that encodes the ligand, a cDNA library is constructed from the isolated stromal cells in a suitable expression vector, preferably a phage such as CDM8, pSV Sport (BRL Gibco) or piH3, (Seed et al., Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987)). The library is transfected into suitable host cells, such as COS cells. Cells containing ligands on their surface are detected by known methods, see above.

In one such method, transfected COS cells are distributed into single cell suspensions and incubated with the secreted alkaline phosphatase-flk receptor fusion protein, which is present in the medium from NIH/3T3 or COS cells prepared by the method described by Flanagan et al., see above. Alkaline phosphatase-receptor fusion proteins that are not bound to the cells are removed by centrifugation, and the cells are panned on plates coated with antibodies to alkaline phosphatase. Bound cells are isolated following several washes with a suitable wash reagent, such as 5% fetal bovine serum in PBS, and the DNA is extracted from the cells. Additional details of the panning method described above may be found in an article by Seed et al., Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987).

In a second strategy, the putative extracellular ligand binding domains of the receptors are fused to the transmembrane and kinase domains of the human c-fms tyrosine kinase and introduced into 3T3 fibroblasts. The human c-fms kinase is necessary and sufficient to transduce proliferative signals in these cells after appropriate activation i.e. with the flk-1 or flk-2 ligand. The 3T3 cells expressing the chimeras are used to screen putative sources of ligand in a cell proliferation assay.

An alternate approach for isolating ligands using the fusion receptor-expressing 3T3 cells and insertional activation is also possible. A retrovirus is introduced into random chromosomal positions in a large population of these cells. In a small fraction, the retrovirus is inserted in the vicinity of the ligand-encoding gene, thereby activating it. These cells proliferate due to autocrine stimulation of the receptor. The ligand gene is "tagged" by the retrovirus, thus facilitating its isolation.

Examples Example 1. Cells containing mouse flk-1 and flk-2 ligands. Murine stromal cell line 2018.

In order to establish stromal cell lines, fetal liver cells are disaggregated with collagen and grown in a mixture of Dulbecco's Modified Eagle's Medium (DMEM) and 10% heat-inactivated fetal calf serum at 37° C. The cells are immortalized by standard methods. A suitable method involves introducing DNA encoding a growth regulating- or oncogene-encoding sequence into the target host cell. The DNA may be introduced by means of transduction in a recombinant viral particle or transfection in a plasmid. See, for example, Hammerschmidt et al., Nature 340, 393-397 (1989) and Abcouwer et al, Biotechnology 7, 939-946 (1989). Retroviruses are the preferred viral vectors, although SV40 and Epstein-Barr virus can also serve as donors of the growth-enhancing sequences. A suitable retrovirus is the ecotropic retrovirus containing a temperature sensitive SV40 T-antigen (tsA58) and a G418 resistance gene described by McKay in Cell 66, 713-729 (1991). After several days at 37° C., the temperature of the medium is lowered to 32° C. Cells are selected with G418 (0.5 mg/ml). The selected cells are expanded and maintained.

A mouse stromal cell line produced by this procedure is called 2018 and was deposited on Oct. 30, 1991 in the American Type Culture Collection, Rockville, Md., USA (ATCC); accession number CRL 10907.

Example 2. Cells containing human flk-1 and flk-2 ligands.

Human fetal liver (18, 20, and 33 weeks after abortion), spleen (18 weeks after abortion), or thymus (20 weeks after abortion) is removed at the time of abortion and stored on ice in a balanced salt solution. After mincing into 1 mm fragments and forcing through a wire mesh, the tissue is washed one time in Hanks Balanced Salt Solution (HBSS).

The disrupted tissue is centrifuged at 200 xg for 15 minutes at room temperature. The resulting pellet is resuspended in 10-20 ml of a tissue culture grade trypsin-EDTA solution (Flow Laboratories). The resuspended tissue is transferred to a sterile flask and stirred with a stirring bar at room temperature for 10 minutes. One ml of heat-inactivated fetal bovine calf serum (Hyclone) is added to a final concentration of 10% in order to inhibit trypsin activity. Collagenase type IV (Sigma) is added from a stock solution (10 mg/ml in HBSS) to a final concentration of 100 ug/ml in order to disrupt the stromal cells. The tissue is stirred at room temperature for an additional 2.5 hours; collected by centrifugation (400xg, 15 minutes); and resuspended in "stromal medium," which contains Iscove's modification of DMEM supplemented with 10% heat-inactivated fetal calf serum, 5% heat-inactivated human serum (Sigma), 4 mM L-glutamine, 1x sodium pyruvate, (stock of 100x Sigma), 1x non-essential amino acids (stock of 100x, Flow), and a mixture of antibiotics kanomycin, neomycin, penicillin, streptomycin. Prior to resuspending the pellet in the stromal medium, the pellet is washed one time with HBSS. It is convenient to suspend the cells in 60 ml of medium. The number of cultures depends on the amount of tissue.

Example 3. Isolating Stromal cells

Resuspended Cells (example 2) that are incubated at 37° C. with 5% carbon dioxide begin to adhere to the plastic plate within 10-48 hours. Confluent monolayers may be observed within 7-10 days, depending upon the number of cells plated in the initial innoculum. Non-adherent and highly refractile cells adhering to the stromal cell layer as colonies are separately removed by pipetting and frozen. Non-adherent cells are likely sources of populations of self-renewing stem cells containing flk-2. The adherent stromal cell layers are frozen in aliquots for future studies or expanded for growth in culture.

An unexpectedly high level of APtag-flk-2 fusion protein binding to the fetal spleen cells is observed. Two fetal spleen lines are grown in "stromal medium," which is described in example 2.

Non-adherent fetal stem cells attach to the stromal cells and form colonies (colony forming unit - CFU). Stromal cells and CFU are isolated by means of sterile glass cylinders and expanded in culture. A clone, called Fsp 62891, contains the flk-2 ligand. Fsp 62891 was deposited in the American Type Culture Collection, Rockville, Md., U.S.A on Nov. 21, 1991, accession number CRL 10935.

Fetal liver and fetal thymus cells are prepared in a similar way. Both of these cell types produce ligands of flk-1 and, in the case of liver, some flk-2. One such fetal thymus cell line, called F.thy 62891, was deposited in the American Type Culture Collection, Rockville, Md., U.S.A on Nov. 21, 1991, accession number CRL 10936.

Human cell lines are prepared from fetal cells with the same temperature sensitive immortalizing virus used to prepare the murine cell line described in example 1.

Example 4. Isolation of human stromal cell clone

Highly refractile cells overgrow patches of stromal cells, presumably because the stromal cells produce factors that allow the formation of the CFU. To isolate stromal cell clones, sterile glass cylinders coated with vacuum grease are positioned over the CFU. A trypsin-EDTA solution (100 ml) is added in order to detach the cells. The cells are added to 5 ml of stromal medium and each (clone) plated in a single well of 6-well plate.

Example 5. Plasmid (AP-tag) for expressing secretable alkaline phosphatase (SEAP)

Plasmids that express secretable alkaline phosphatase are described by Flanagan and Leder in Cell 63, 185-194 (1990). The plasmids contain a promoter, such as the LTR promoter; a polylinker, including HindIII and BglII; DNA encoding SEAP; a poly-A signal; and ampicillin resistance gene; and replication site.

Example 6. Plasmid for expressing APtag-flk-2 and APtag-flk-1 fusion proteins

Plasmids that express fusion proteins of SEAP and the extracellular portion of either flk-1 or flk-2 are prepared in accordance with the protocols of Flanagan and

Leder in Cell 63, 185-194 (1990) and Berger et al., Gene 66, 1-10 (1988). Briefly, a HindIIIBam HI fragment containing the extracellular portion of flk-1 or flk-2 is prepared and inserted into the HindIII-BglII site of the plasmid described in example 5.

Example 7. Production Of APtag-flk-1 Or -flk-2 Fusion Protein

The plasmids from Example 6 are transfected into Cos-7 cells by DEAE-dextran (as described in Current Protocols in Molecular Biology, Unit 16.13, "Transient Expression of Proteins Using Cos Cells," 1991); and cotransfected with a selectable marker, such as pSV7neo, into NIH/3T3 cells by calcium precipitation. The NIH/3T3 cells are selected with 600μg/ml G418 in 100 mm plates. Over 300 clones are screened for secretion of placental alkaline phosphatase activity. The assay is performed by heating a portion of the supernatant at 65° C. for 10 minutes to inactivate background phosphatase activity, and measuring the OD₄₀₅ after incubating with 1M diethanolamine (pH 9.8), 0.5 mM MgCl₂, 10 mM L-homoarginine (a phosphatase inhibitor), 0.5 mg/ml BSA, and 12 mM p-nitrophenyl phosphate. Human placental alkaline phosphatase is used to perform a standard curve. The APtag-flk-1 clones (F-1AP21-4) produce up to 10 μg alkaline phosphatase activity/ml and the APtag-flk-2 clones (F-2AP26-0) produce up to 0.5 μg alkaline phosphatase activity/ml.

Example 8. Assay For APtag-flk-1 Or APtag-flk-2 Binding To Cells

The binding of APtag-flk-1 or APtag-flk-2 to cells containing the appropriate ligand is assayed by standard methods. See, for example, Flanagan and Leder, Cell 63:185-194, 1990). Cells (i.e., mouse stromal cells, human fetal liver, spleen or thymus, or various control cells) are grown to confluency in six-well plates and washed with HBHA (Hank's balanced salt solution with 0.5 mg/ml BSA, 0.02% NaN₃, 20 mM HEPES, pH 7.0). Supernatants from transfected COS or NIH/3T3 cells containing either APtag-flk-1 fusion protein, APtag-flk-2 fusion protein, or APtag without a receptor (as a control) are added to the cell monolayers and incubated for two hours at room temperature on a rotating platform. The concentration of the APtag-flk-1 fusion protein, APtag-flk-2 fusion protein, or APtag without a receptor is 60 ng/ml of alkaline phosphatase as determined by the standard alkaline phosphatase curve (see above). The cells are then rinsed seven times with HBHA and lysed in 350 μl of 1% Triton X-100, 10 mM Tris-HCl (pH 8.0). The lysates re transferred to a microfuge tube, along with a further 150 μl rinse with the same solution. After vortexing vigorously, the samples are centrifuged for five minutes in a microfuge, heated at 65° C. for 12 minutes to inactivate cellular phosphatases, and assayed for phosphatase activity as described previously. Results of experiments designed to show the time and dose responses of binding between stromal cells containing the ligands to flk-2 and flk-1 (2018) and APtag-flk-2, APtag-flk-1 and APtag without receptor (as a control) are shown in FIGS. 3 and 4, respectively.

Example 9. Cloning and Expression of cDNA Coding For Mouse Ligand To flk-1 and flk-2 Receptors

cDNA expressing mouse ligand for flk-1 and flk-2 is prepared by known methods. See, for example, Seed, B., and Aruffo, A. PNAS 84:3365-3369, 1987; Simmons, D. and Seed, B. J. Immunol. 141:2797-2800; and D'Andrea, A. D., Lodish, H. F. and Wong, G. G. Cell 57:277-285, 1989).

The protocols are listed below in sequence: (a) RNA isolation; (b) poly A RNA preparation; (c) cDNA synthesis; (d) cDNA size fractionation; (e) propagation of plasmids (vector); (f) isolation of plasmid DNA; (g) preparation of vector pSV Sport (BRL Gibco) for cloning; (h) compilation of buffers for the above steps; (i) Transfection of cDNA encoding Ligands in Cos 7 Cells; (j) panning procedure.

9a. Guanidinium thiocyanate/LiCl Protocol for RNA Isolation

For each ml of mix desired, 0.5 g guanidine thiocyanate (GuSCN) is dissolved in 0.55 ml of 25% LiCl (stock filtered through 0.45 micron filter). 20 μl of mercaptoethanol is added. (The resulting solution is not good for more than about a week at room temperature.)

The 2018 cells are centrifuged, and 1 ml of the solution described above is added to up to 5×10⁷ cells. The cells are sheared by means of a polytron until the mixture is non-viscous. For small scale preparations (<10⁸ cells), the sheared mixture is layered on 1.5 ml of 5.7M CsCl (RNase free; 1.26 g CsCl added to every ml 10 mM EDTA pH8), and overlaid with RNase-free water if needed. The mixture is spun in an SW55 rotor at 50 krpm for 2 hours. For large scale preparations, 25 ml of the mixture is layered on 12 ml CsCl in an SW28 tube, overlaid as above, and spun at 24 krpm for 8 hours. The contents of the tube are aspirated carefully with a sterile pasteur pipet connected to a vacuum flask. Once past the CsCl interface, a band around the tube is scratched with the pipet tip to prevent creeping of the layer on the wall down the tube. The remaining CsCl solution is aspirated. The resulting pellet is taken up in water, but not redissolved. 1/10 volume of sodium acetate and three volumes of ethanol are added to the mixture, and spun. The pellet is resuspended in water at 70o, if necessary. The concentration of the RNA is adjusted to 1 mg/ml and frozen.

It should be noted that small RNA molecules (e.g., 5S) do not come down. For small amounts of cells, the volumes are scaled down, and the mixture is overlaid with GuSCN in RNase-free water on a gradient (precipitation is inefficient when RNA is dilute).

9b. Poly A RNA preparation

(All buffers mentioned are compiled separately below)

A disposable polypropylene column is prepared by washing with 5M NaOH and then rinsing with RNase-free water. For each milligram of total RNA, approximately 0.3 ml (final packed bed) of oligo dT cellulose is added. The oligo dT cellulose is prepared by resuspending approximately 0.5 ml of dry powder in 1 ml of 0.1M NaOH and transferring it into the column, or by percolating 0.1M NaOH through a previously used column. The column is washed with several column volumes of RNase-free water until the pH is neutral, and rinsed with 2-3 ml of loading buffer. The column bed is transferred to a sterile 15 ml tube using 4-6 ml of loading buffer.

Total RNA from the 2018 cell line is heated to 70° C. for 2-3 minutes. LiCl from RNase-free stock is added to the mixture to a final concentration of 0.5M. The mixture is combined with oligo dT cellulose in the 15 ml tube, which is vortexed or agitated for 10 minutes. The mixture is poured into the column, and washed with 3 ml loading buffer, and then with 3 ml of middle wash buffer. The mRNA is eluted directly into an SW55 tube with 1.5 ml of 2 mM EDTA and 0.1% SDS, discarding the first two or three drops.

The eluted mRNA is precipitated by adding 1/10 volume of 3M sodium acetate and filling the tube with ethanol. The contents of the tube are mixed, chilled for 30 minutes at -20°, and spun at 50 krpm at 5° C. for 30 minutes. After the ethanol is decanted, and the tube air dried, the mRNA pellet is resuspended in 50-100 μl of RNase-free water. 5 of the resuspended mRNA is heated to 70° C. in MOPS/EDTA/formaldehyde, and examined on an RNase-free 1% agarose gel.

9c. cDNA Synthesis

The protocol used is a variation of the method described by Gubler and Hoffman in Gene 25, 263-270 (1983).

1. First Strand. 4 μg of mRNA is added to a microfuge tube, heated to approximately 100° C. for 30 seconds, quenched on ice. The volume is adjusted to 70μl with RNAse-free water. 20 μl of RT1 buffer, 2 μl of RNAse inhibitor (Boehringer 36 u/μl ), 1 μl of 5 μg/μl of oligo dT (Collaborative Research), 2.5 μl of 20 mM dXTP's (ultrapure - US Biochemicals), 1 μl of 1M DTT and 4 μl of RT-XL (Life Sciences, 24 u/μl ) are added. The mixture is incubated at 42° C. for 40 minutes, and inactivated by heating at 70° C. for 10 minutes.

2. Second Strand. 320 μl of RNAse-free water, 80 μl of RT2 buffer, 5 μl of DNA Polymerase I (Boehringer, 5 U/μl), 2 μl RNAse H (BRL 2 u/μl) are added to the solution containing the first strand. The solution is incubated at 15° C. for one hour and at 22° C. for an additional hour. After adding 20 μl of 0.5M EDTA, pH 8.0, the solution is extracted with phenol and precipitated by adding NaCl to 0.5M linear polyacrylamide (carrier) to 20 μg/ml, and filling the tube with EtOH. The tube is spun for 2-3 minutes in a microfuge, vortexed to dislodge precipitated material from the wall of the tube, and respun for one minute.

3. Adaptors. Adaptors provide specific restriction sites to facilitate cloning, and are available from BRL Gibco, New England Biolabs, etc. Crude adaptors are resuspended at a concentration of 1 μg/μl. MgSO₄ is added to a final concentration of 10 mM, followed by five volumes of EtOH. The resulting precipitate is rinsed with 70% EtOH and resuspended in TE at a concentration of 1 μg/μl. To kinase, 25 μl of resuspended adaptors is added to 3 μl of 10X kinasing buffer and 20 units of kinase. The mixture is incubated at 37° C. overnight. The precipitated cDNA is resuspended in 240 μl of TE (10/1). After adding 30 μl of 10X low salt buffer, 30 μl of 10X ligation buffer with 0.1 mM ATP, 3 μl (2.4 μg) of kinased 12-mer adaptor sequence, 2 μl (1.6 μg) of kinased 8-mer adaptor sequence, and 1 μl of T4 DNA ligase (BioLabs, 400 u/μl, or Boehringer, 1 Weiss unit ml), the mixture is incubated at 15° C. overnight. The cDNA is extracted with phenol and precipitated as above, except that the extra carrier is omitted, and resuspended in 100 μl of TE.

9d. cDNA Size Fractionation.

A 20% KOAc, 2 mM EDTA, 1 μg/ml ethidium bromide solution and a 5% KOAc, 2 mM EDTA, 1 μg/ml ethidium bromide solution are prepared. 2.6 ml of the 20% KOAc solution is added to the back chamber of a small gradient maker. Air bubbles are removed from the tube connecting the two chambers by allowing the 20% solution to flow into the front chamber and forcing the solution to return to the back chamber by tilting the gradient maker. The passage between the chambers is closed, and 2.5 ml of 5% solution is added to the front chamber. Any liquid in the tubing from a previous run is removed by allowing the 5% solution to flow to the end of the tubing, and then to return to its chamber. The apparatus is placed on a stirplate, and, with rapid stirring, the topcock connecting the two chambers and the front stopcock are opened. A polyallomer 5W55 tube is filled from the bottom with the KOAc solution. The gradient is overlaid with 100 μl of cDNA solution, and spun for three hours at 50 k rpm at 22°. To collect fractions from the gradient, the SW55 tube is pierced close to the bottom of the tube with a butterfly infusion set (with the luer hub clipped off). Three 0.5 ml fractions and then six 0.25 ml fractions are collected in microfuge tubes (approximately 22 and 11 drops, respectively). The fractions are precipitated by adding linear polyacrylamide to 20 μg/ml and filling the tube to the top with ethanol. The tubes are cooled, spun in a microfuge tube for three minutes, vortexed, and respun for one minute. The resulting pellets are rinsed with 70% ethanol and respun, taking care not to permit the pellets to dry to completion. Each 0.25 ml fraction is resuspended in 10 μl of TE, and 1 μl is run on a 1% agarose minigel. The first three fractions, and the last six which contain no material smaller than 1 kb are pooled.

9e. Propagation of Plasmids

SupF plasmids are selected in nonsuppressing bacterial hosts containing a second plasmid, p3, which contains amber mutated ampicillin and tetracycline drug resistance elements. See Seed, Nucleic Acids Res., 11, 2427-2445 (1983). The p3 plasmid is derived from RPI, is 57 kb in length, and is a stably maintained, single copy episome. The ampicillin resistance of this plasmid reverts at a high rate so that amp^(r) plasmids usually cannot be used in p3-containing strains. Selection for tetracycline resistance alone is almost as good as selection for ampicillin-tetracycline resistance. However, spontaneous appearance of chromosomal suppressor tRNA mutations presents an unavoidable background (frequency about 10⁻⁹) in this system. Colonies arising from spontaneous suppressor mutations are usually larger than colonies arising from plasmid transformation. Suppressor plasmids are selected in Luria broth (LB) medium containing ampicillin at 12.5 μg/ml and tetr2acycline at 7.5 μg/ml. For scaled-up plasmid preparations, M9 Casamino acids medium containing glycerol (0.8%) is employed as a carbon source. The bacteria are grown to saturation.

Alternatively, pSV Sport (BRL, Gaithersberg, Md.) may be employed to provide SV40 derived sequences for replication, transcription initiation and termination in COS 7 cells, as well as those sequences necessary for replication and ampicillin resistance in E. coli.

9f. Isolation of Vector DNA/Plasmid

One liter of saturated bacterial cells are spun down in J6 bottles at 4.2k rpm for 25 minutes. The cells are resuspended in 40 ml 10 mM EDTA, pH 8. 80 ml 0.2M NaOH and 1% SDS are added, and the mixture is swirled until it is clear and viscous. 40 ml 5M KOAc, pH 4.7 (2.5M KOAc, 2.5M HOAc) is added, and the mixture is shaken semi-vigorously until the lumps are approximately 2-3 mm in size. The bottle is spun at 4.2 k rpm for 5 minutes. The supernatant is poured through cheesecloth into a 250 ml bottle, which is then filled with isopropyl alcohol and centrifuged at 4.2 k rpm for 5 minutes. The bottle is gently drained and rinsed with 70% ethanol, taking care not to fragment the pellet. After inverting the bottle and removing traces of ethanol, the mixture is resuspended in 3.5 ml Tris base/EDTA (20 mM/10 mM). 3.75 ml of resuspended pellet and 0.75 ml 10 mg/ml ethidium bromide are added to 4.5 g CsCl. VTi80 tubes are filled with solution, and centrifuged for at least 2.5 hours at 80k rpm. Bands are extracted by visible light with 1 ml syringe and 20 gauge or lower needle. The top of the tube is cut off with scissors, and the needle is inserted upwards into the tube at an angle of about 30 degrees with respect to the tube at a position about 3 mm beneath the band, with the bevel of the needle up. After the band is removed, the contents of the tube are poured into bleach. The extracted band is deposited in a 13 ml Sarstedt tube, which is then filled to the top with n-butanol saturated with 1M NaCl extract. If the amount of DNA is large, the extraction procedure may be repeated. After aspirating the butanol into a trap containing 5M NaOH to destroy ethidium, an approximately equal volume of 1M ammonium acetate and approximately two volumes of 95% ethanol are added to the DNA, which is then spun at 10 k rpm for 5 minutes. The pellet is rinsed carefully with 70% ethanol, and dried with a swab or lyophilizer.

9g. Preparation of Vector for Cloning

20 g of vector is cut in a 200 μl reaction with 100 units of BstXI (New York Biolabs) at 50° C. overnight in a well thermostated, circulating water bath. Potassium acetate solutions (5 and 20%) are prepared in 5 W55 tubes as described above. 100 μl of the digested vector is added to each tube and spun for three hours, 50 k rpm at 22° C. Under 300 nm UV light, the desired band is observed to migrate 2/3 of the length of the tube. Forward trailing of the band indicates that the gradient is overloaded. The band is removed with a 1 ml syringe fitted with a 20 gauge needle. After adding linear polyacrylamide and precipitating the plasmid by adding three volumes of ethanol, the plasmid is resuspended in 50 μl of TE. Trial ligations are carried out with a constant amount of vector and increasing amounts of cDNA. Large scale ligation are carried out on the basis of these trial ligations. Usually the entire cDNA prep requires 1-2 μg of cut vector.

    ______________________________________                                         9h. Buffers                                                                    ______________________________________                                         Loading Buffer:                                                                              .5M LiCl, 10 mM Tris pH 7.5, 1 mM                                              EDTA .1% SDS.                                                    Middle Wash Buffer:                                                                          .15M LiCl, 10 mM Tris pH 7.5, 1 mM                                             EDTA .1% SDS.                                                    RT1 Buffer:   .25M Tris pH 8.8 (8.2 at 42.sup.-, .25M                                        KCl, 30 mM MgCl.sub.2.                                           RT2 Buffer:   .1M Tris pH 7.5, 25 mM MgCl2, .5M                                              KCl, .25 mg/ml BSA, 50 mM                                                      dithiothreitol (DTT).                                            10X Low Salt: 60 mM Tris pH 7.5, 60 mM MgCl.sub.2, 50                                        mM NaCl, 2.5 mg/ml BSA 70 mM                                                   DME                                                              10X Ligation Additions:                                                                      1 mM ATP, 20 mM DTT, 1 mg/ml                                                   BSA 10 mM spermidine.                                            10X Kinasing Buffer:                                                                         .5M Tris pH 7.5, 10 mM ATP, 20 mM                                              DTT, 10 mM spermidine, 1 mg/ml BSA                                             100 mM MgCl.sub.2                                                ______________________________________                                    

9i. Transfection of cDNA encoding Ligands in Cos 7 Cells

Cos 7 cells are split 1:5 into 100 mm plates in Dulbecco's modified Eagles medium (DME)/10% fetal calf serum (FCS), and allowed to grow overnight. 3 ml Tris/DME (0.039M Tris, pH 7.4 in DME) containing 400 μg/ml DEAE-dextran (Sigman, D-9885) is prepared for each 100 mm plate of Cos 7 cells to be transfected. 10 ug of plasmid DNA preparation per plate is added. The medium is removed from the Cos-7 cells and the DNA/DEAE-dextran mixture is added. The cells are incubated for 4.5 hours. The medium is removed from the cells, and replaced with 3 ml of DME containing 2% fetal calf serum (FCS) and 0.1 mM chloroquine. The cells are incubated for one hour. After removing the chloroquine and replacing with 1.5 ml 20% glycerol in PBS, the cells are allowed to stand at room temperature for one minute. 3 ml Tris/DME is added, and the mixture is aspirated and washed two times with Tris/DME. 10 ml DME/10% FCS is added and the mixture is incubated overnight. The transfected Cos 7 cells are split 1:2 into fresh 100 mm plates with (DME)/10% FCS and allowed to grow.

9j. Panning Procedure for Cos 7 cells Expressing Ligand

1) Antibody-coated plates:

Bacteriological 100 mm plates are coated for 1.5 hours with rabbit anti-human placental alkaline phosphatase (Dako, Calif.) diluted 1:500 in 10 ml of 50 mM Tris.HCl, pH 9.5. The plates are washed three times with 0.15M NaCl, and incubated with 3 mg BSA/ml PBS overnight. The blocking solution is aspirated, and the plates are utilized immediately or frozen for later use.

2) Panning cells:

The medium from transfected Cos 7 cells is aspirated, and 3 ml PBS/0.5 mM EDTA/0.02% sodium azide is added. The plates are incubated at 37° C. for thirty minutes in order to detach the cells. The cells are triturated vigorously with a pasteur pipet and collected in a 15 ml centrifuge tube. The plate is washed with a further 2 ml PBS/EDTA/azide solution, which is then added to the centrifuge tube. After centrifuging at 200 xg for five minutes, the cells are resuspended in 3 ml of APtaq-flk-1 (F-1AP21-4) or flk-2 (F-2AP26-0) supernatant from transfected NIH/3T3 cells (see Example 7.), and incubated for 1.5 hours on ice. The cells are centrifuged again at 200 xg for five minutes. The supernatant is aspirated, and the cells are resuspended in 3 ml PBS/EDTA/azide solution. The cell suspension is layered carefully on 3 ml PBS/EDTA/azide/2% Ficoll, and centrifuged at 200 xg for four minutes. The supernatant is aspirated, and the cells are resuspended in 0.5 ml PBS/EDTA/azide solution. The cells are added to the antibody-coated plates containing 4 ml PBS/EDTA/azide/5% FBS, and allowed to stand at room temperature one to three hours. Non-adhering cells are removed by washing gently two or three times with 3 ml PBS/5% FBS.

3) Hirt Supernatant:

0.4 ml 0.6% SDS and 10 mM EDTA are added to the panned plates, which are allowed to stand 20 minutes. The viscuous mixture is added by means of a pipet into a microfuge tube. 0.1ml 5M NaCl is added to the tube, mixed, and chilled on ice for at least five hours. The tube is spun for four minutes, and the supernatant is removed carefully. The contents of the tube are extracted with phenol once, or, if the first interface is not clean, twice. Ten micrograms of linear polyacrylamide (or other carrier) is added, and the tube is filled to the top with ethanol. The resulting precipitate is resuspended in 0.1 ml water or TE. After adding 3 volumes of EtOH/NaOAc, the cells are reprecipitated and resuspended in 0.1 ml water or TE. The cDNA obtained is transfected into any suitable E. coli host by electroporation. Suitable hosts are described in various catalogs, and include MC1061/p3 or Electromax DH10B Cells of BRL Gibco. The cDNA is extracted by conventional methods.

The above panning procedure is repeated until a pure E. coli clone bearing the cDNA as a unique plasmid recombinant capable of transfecting mammalian cells and yielding a positive panning assay is isolated. Normally, three repetitions are sufficient.

Example 10. Expression of Ligand cDNA

The cDNA is sequenced, and expressed in a suitable host cell, such as a mammalian cell, preferably COS, CHO or NIH/3T3 cells. The presence of the ligand is confirmed by demonstrating binding of the ligand to APtag-flk2 fusion protein (see above).

SUPPLEMENTAL ENABLEMENT

The invention as claimed is enabled in accordance with the above specification and readily available references and starting materials. Nevertheless, on Oct. 30, 1991, Applicants deposited with the American Type Culture Collection, Rockville, Md., USA (ATCC) the cell lines listed below:

2018, ATCC accession no. CRL 10907.

Fsp 62891, ATCC accession no. CRL 10935

F.thy 62891, ATCC accession no. CRL 10936

These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty). This assures maintenance of a visible culture for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 4                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3453 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 31..3009                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: matpeptide                                                       (B) LOCATION: 31..3006                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GCGGCCTGGCTACCGCGCGCTCCGGAGGCCATGCGGGCGTTGGCGCAGCGCAGC54                       MetArgA laLeuAlaGlnArgSer                                                      15                                                                             GACCGGCGGCTGCTGCTGCTTGTTGTTTTGTCAGTAATGATTCTTGAG102                            AspArgArgLeuLeuLeuLeuValValLeuSerVal MetIleLeuGlu                              101520                                                                         ACCGTTACAAACCAAGACCTGCCTGTGATCAAGTGTGTTTTAATCAGT150                            ThrValThrAsnGlnAspLeuProValIleLysCysValLeuIle Ser                              25303540                                                                       CATGAGAACAATGGCTCATCAGCGGGAAAGCCATCATCGTACCGAATG198                            HisGluAsnAsnGlySerSerAlaGlyLysProSerSerTyr ArgMet                              455055                                                                         GTGCGAGGATCCCCAGAAGACCTCCAGTGTACCCCGAGGCGCCAGAGT246                            ValArgGlySerProGluAspLeuGlnCysThrProArgArg GlnSer                              606570                                                                         GAAGGGACGGTATATGAAGCGGCCACCGTGGAGGTGGCCGAGTCTGGG294                            GluGlyThrValTyrGluAlaAlaThrValGluValAlaGluSer Gly                              758085                                                                         TCCATCACCCTGCAAGTGCAGCTCGCCACCCCAGGGGACCTTTCCTGC342                            SerIleThrLeuGlnValGlnLeuAlaThrProGlyAspLeuSerCys                                9095100                                                                       CTCTGGGTCTTTAAGCACAGCTCCCTGGGCTGCCAGCCGCACTTTGAT390                            LeuTrpValPheLysHisSerSerLeuGlyCysGlnProHisPheAsp                               105 110115120                                                                  TTACAAAACAGAGGAATCGTTTCCATGGCCATCTTGAACGTGACAGAG438                            LeuGlnAsnArgGlyIleValSerMetAlaIleLeuAsnValThrGlu                                125130135                                                                     ACCCAGGCAGGAGAATACCTACTCCATATTCAGAGCGAACGCGCCAAC486                            ThrGlnAlaGlyGluTyrLeuLeuHisIleGlnSerGluArgAlaAsn                                140145150                                                                     TACACAGTACTGTTCACAGTGAATGTAAGAGATACACAGCTGTATGTG534                            TyrThrValLeuPheThrValAsnValArgAspThrGlnLeuTyrVal                               155 160165                                                                     CTAAGGAGACCTTACTTTAGGAAGATGGAAAACCAGGATGCACTGCTC582                            LeuArgArgProTyrPheArgLysMetGluAsnGlnAspAlaLeuLeu                               170 175180                                                                     TGCATCTCCGAGGGTGTTCCGGAGCCCACTGTGGAGTGGGTGCTCTGC630                            CysIleSerGluGlyValProGluProThrValGluTrpValLeuCys                               185190 195200                                                                  AGCTCCCACAGGGAAAGCTGTAAAGAAGAAGGCCCTGCTGTTGTCAGA678                            SerSerHisArgGluSerCysLysGluGluGlyProAlaValValArg                               205 210215                                                                     AAGGAGGAAAAGGTACTTCATGAGTTGTTCGGAACAGACATCAGATGC726                            LysGluGluLysValLeuHisGluLeuPheGlyThrAspIleArgCys                               220 225230                                                                     TGTGCTAGAAATGCACTGGGCCGCGAATGCACCAAGCTGTTCACCATA774                            CysAlaArgAsnAlaLeuGlyArgGluCysThrLysLeuPheThrIle                               235240 245                                                                     GATCTAAACCAGGCTCCTCAGAGCACACTGCCCCAGTTATTCCTGAAA822                            AspLeuAsnGlnAlaProGlnSerThrLeuProGlnLeuPheLeuLys                               250255 260                                                                     GTGGGGGAACCCTTGTGGATCAGGTGTAAGGCCATCCATGTGAACCAT870                            ValGlyGluProLeuTrpIleArgCysLysAlaIleHisValAsnHis                               265270275 280                                                                  GGATTCGGGCTCACCTGGGAGCTGGAAGACAAAGCCCTGGAGGAGGGC918                            GlyPheGlyLeuThrTrpGluLeuGluAspLysAlaLeuGluGluGly                               285290 295                                                                     AGCTACTTTGAGATGAGTACCTACTCCACAAACAGGACCATGATTCGG966                            SerTyrPheGluMetSerThrTyrSerThrAsnArgThrMetIleArg                               300305 310                                                                     ATTCTCTTGGCCTTTGTGTCTTCCGTGGGAAGGAACGACACCGGATAT1014                           IleLeuLeuAlaPheValSerSerValGlyArgAsnAspThrGlyTyr                               315320325                                                                      T ACACCTGCTCTTCCTCAAAGCACCCCAGCCAGTCAGCGTTGGTGACC1062                          TyrThrCysSerSerSerLysHisProSerGlnSerAlaLeuValThr                               330335340                                                                      ATCCTAGAAA AAGGGTTTATAAACGCTACCAGCTCGCAAGAAGAGTAT1110                          IleLeuGluLysGlyPheIleAsnAlaThrSerSerGlnGluGluTyr                               345350355360                                                                   GAAATTG ACCCGTACGAAAAGTTCTGCTTCTCAGTCAGGTTTAAAGCG1158                          GluIleAspProTyrGluLysPheCysPheSerValArgPheLysAla                               365370375                                                                      TACCCAC GAATCCGATGCACGTGGATCTTCTCTCAAGCCTCATTTCCT1206                          TyrProArgIleArgCysThrTrpIlePheSerGlnAlaSerPhePro                               380385390                                                                      TGTGAACAGA GAGGCCTGGAGGATGGGTACAGCATATCTAAATTTTGC1254                          CysGluGlnArgGlyLeuGluAspGlyTyrSerIleSerLysPheCys                               395400405                                                                      GATCATAAGAACAAGC CAGGAGAGTACATATTCTATGCAGAAAATGAT1302                          AspHisLysAsnLysProGlyGluTyrIlePheTyrAlaGluAsnAsp                               410415420                                                                      GACGCCCAGTTCACCAAAATGTTCA CGCTGAATATAAGAAAGAAACCT1350                          AspAlaGlnPheThrLysMetPheThrLeuAsnIleArgLysLysPro                               425430435440                                                                   CAAGTGCTAGCAAATGCCTCAG CCAGCCAGGCGTCCTGTTCCTCTGAT1398                          GlnValLeuAlaAsnAlaSerAlaSerGlnAlaSerCysSerSerAsp                               445450455                                                                      GGCTACCCGCTACCCTCTTGGA CCTGGAAGAAGTGTTCGGACAAATCT1446                          GlyTyrProLeuProSerTrpThrTrpLysLysCysSerAspLysSer                               460465470                                                                      CCCAATTGCACGGAGGAAATCCCAG AAGGAGTTTGGAATAAAAAGGCT1494                          ProAsnCysThrGluGluIleProGluGlyValTrpAsnLysLysAla                               475480485                                                                      AACAGAAAAGTGTTTGGCCAGTGGGTGTCGA GCAGTACTCTAAATATG1542                          AsnArgLysValPheGlyGlnTrpValSerSerSerThrLeuAsnMet                               490495500                                                                      AGTGAGGCCGGGAAAGGGCTTCTGGTCAAATGCTGTGCGT ACAATTCT1590                          SerGluAlaGlyLysGlyLeuLeuValLysCysCysAlaTyrAsnSer                               505510515520                                                                   ATGGGCACGTCTTGCGAAACCATCTTTTTAAACTCAC CAGGCCCCTTC1638                          MetGlyThrSerCysGluThrIlePheLeuAsnSerProGlyProPhe                               525530535                                                                      CCTTTCATCCAAGACAACATCTCCTTCTATGCGACCA TTGGGCTCTGT1686                          ProPheIleGlnAspAsnIleSerPheTyrAlaThrIleGlyLeuCys                               540545550                                                                      CTCCCCTTCATTGTTGTTCTCATTGTGTTGATCTGCCACA AATACAAA1734                          LeuProPheIleValValLeuIleValLeuIleCysHisLysTyrLys                               555560565                                                                      AAGCAATTTAGGTACGAGAGTCAGCTGCAGATGATCCAGGTGACTG GC1782                          LysGlnPheArgTyrGluSerGlnLeuGlnMetIleGlnValThrGly                               570575580                                                                      CCCCTGGATAACGAGTACTTCTACGTTGACTTCAGGGACTATGAATAT1830                            ProLeuAspAsnGluTyrPheTyrValAspPheArgAspTyrGluTyr                              585590595600                                                                   GACCTTAAGTGGGAGTTCCCGAGAGAGAACTTAGAGTTTGGGAAGGTC18 78                          AspLeuLysTrpGluPheProArgGluAsnLeuGluPheGlyLysVal                               605610615                                                                      CTGGGGTCTGGCGCTTTCGGGAGGGTGATGAACGCCACGGCCTATGGC19 26                          LeuGlySerGlyAlaPheGlyArgValMetAsnAlaThrAlaTyrGly                               620625630                                                                      ATTAGTAAAACGGGAGTCTCAATTCAGGTGGCGGTGAAGATGCTAAAA1974                            IleSerLysThrGlyValSerIleGlnValAlaValLysMetLeuLys                              635640645                                                                      GAGAAAGCTGACAGCTGTGAAAAAGAAGCTCTCATGTCGGAGCTCAAA2022                           GluLys AlaAspSerCysGluLysGluAlaLeuMetSerGluLeuLys                              650655660                                                                      ATGATGACCCACCTGGGACACCATGACAACATCGTGAATCTGCTGGGG2070                           MetMetThrHisLeu GlyHisHisAspAsnIleValAsnLeuLeuGly                              665670675680                                                                   GCATGCACACTGTCAGGGCCAGTGTACTTGATTTTTGAATATTGTTGC2118                           AlaCysThrLeu SerGlyProValTyrLeuIlePheGluTyrCysCys                              685690695                                                                      TATGGTGACCTCCTCAACTACCTAAGAAGTAAAAGAGAGAAGTTTCAC2166                           TyrGlyAspLeu LeuAsnTyrLeuArgSerLysArgGluLysPheHis                              700705710                                                                      AGGACATGGACAGAGATTTTTAAGGAACATAATTTCAGTTCTTACCCT2214                           ArgThrTrpThrGlu IlePheLysGluHisAsnPheSerSerTyrPro                              715720725                                                                      ACTTTCCAGGCACATTCAAATTCCAGCATGCCTGGTTCACGAGAAGTT2262                           ThrPheGlnAlaHisSerAsn SerSerMetProGlySerArgGluVal                              730735740                                                                      CAGTTACACCCGCCCTTGGATCAGCTCTCAGGGTTCAATGGGAATTCA2310                           GlnLeuHisProProLeuAspGlnLeuSer GlyPheAsnGlyAsnSer                              745750755760                                                                   ATTCATTCTGAAGATGAGATTGAATATGAAAACCAGAAGAGGCTGGCA2358                           IleHisSerGluAspGluIleGluTyr GluAsnGlnLysArgLeuAla                              765770775                                                                      GAAGAAGAGGAGGAAGATTTGAACGTGCTGACGTTTGAAGACCTCCTT2406                           GluGluGluGluGluAspLeuAsnVal LeuThrPheGluAspLeuLeu                              780785790                                                                      TGCTTTGCGTACCAAGTGGCCAAAGGCATGGAATTCCTGGAGTTCAAG2454                           CysPheAlaTyrGlnValAlaLysGlyMet GluPheLeuGluPheLys                              795800805                                                                      TCGTGTGTCCACAGAGACCTGGCAGCCAGGAATGTGTTGGTCACCCAC2502                           SerCysValHisArgAspLeuAlaAlaArgAsnVal LeuValThrHis                              810815820                                                                      GGGAAGGTGGTGAAGATCTGTGACTTTGGACTGGCCCGAGACATCCTG2550                           GlyLysValValLysIleCysAspPheGlyLeuAlaArgAspIle Leu                              825830835840                                                                   AGCGACTCCAGCTACGTCGTCAGGGGCAACGCACGGCTGCCGGTGAAG2598                           SerAspSerSerTyrValValArgGlyAsnAlaArgLeuPro ValLys                              845850855                                                                      TGGATGGCACCCGAGAGCTTATTTGAAGGGATCTACACAATCAAGAGT2646                           TrpMetAlaProGluSerLeuPheGluGlyIleTyrThrIle LysSer                              860865870                                                                      GACGTCTGGTCCTACGGCATCCTTCTCTGGGAGATATTTTCACTGGGT2694                           AspValTrpSerTyrGlyIleLeuLeuTrpGluIlePheSerLeu Gly                              875880885                                                                      GTGAACCCTTACCCTGGCATTCCTGTCGACGCTAACTTCTATAAACTG2742                           ValAsnProTyrProGlyIleProValAspAlaAsnPheTyrLysLeu                                890895900                                                                     ATTCAGAGTGGATTTAAAATGGAGCAGCCATTCTATGCCACAGAAGGG2790                           IleGlnSerGlyPheLysMetGluGlnProPheTyrAlaThrGluGly                               905 910915920                                                                  ATATACTTTGTAATGCAATCCTGCTGGGCTTTTGACTCAAGGAAGCGG2838                           IleTyrPheValMetGlnSerCysTrpAlaPheAspSerArgLysArg                                925930935                                                                     CCATCCTTCCCCAACCTGACTTCATTTTTAGGATGTCAGCTGGCAGAG2886                           ProSerPheProAsnLeuThrSerPheLeuGlyCysGlnLeuAlaGlu                                940945950                                                                     GCAGAAGAAGCATGTATCAGAACATCCATCCATCTACCAAAACAGGCG2934                           AlaGluGluAlaCysIleArgThrSerIleHisLeuProLysGlnAla                               955 960965                                                                     GCCCCTCAGCAGAGAGGCGGGCTCAGAGCCCAGTCGCCACAGCGCCAG2982                           AlaProGlnGlnArgGlyGlyLeuArgAlaGlnSerProGlnArgGln                               970 975980                                                                     GTGAAGATTCACAGAGAAAGAAGTTAGCGAGGAGGCCTTGGACCCCGCCACCCT3036                     ValLysIleHisArgGluArgSer                                                       985990                                                                         AGCAGGCTGTAGACCGCAGAGCCAAGATTAGCCTCGCC TCTGAGGAAGCGCCCTACAGCG3096              CGTTGCTTCGCTGGACTTTTCTCTAGATGCTGTCTGCCATTACTCCAAAGTGACTTCTAT3156               AAAATCAAACCTCTCCTCGCACAGGCGGGAGAGCCAATAATGAGACTTGTTGGTGAGCCC3216               GCCTACCCTGGGGGCCTT TCCACGAGCTTGAGGGGAAAGCCATGTATCTGAAATATAGTA3276              TATTCTTGTAAATACGTGAAACAAACCAAACCCGTTTTTTGCTAAGGGAAAGCTAAATAT3336               GATTTTTAAAAATCTATGTTTTAAAATACTATGTAACTTTTTCATCTATTTAGTGATATA3396               TTTTATGGATGGAAATAAACTTTCTACTGTAAAAAAAAAAAAAAAAAAAAAAAAAAA3453                  (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 992 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetArgAlaLe uAlaGlnArgSerAspArgArgLeuLeuLeuLeuVal                              151015                                                                         ValLeuSerValMetIleLeuGluThrValThrAsnGlnAspLeuPro                               20 2530                                                                        ValIleLysCysValLeuIleSerHisGluAsnAsnGlySerSerAla                               354045                                                                         GlyLysProSerSerTyrArgMetValArgGlyS erProGluAspLeu                              505560                                                                         GlnCysThrProArgArgGlnSerGluGlyThrValTyrGluAlaAla                               65707580                                                                        ThrValGluValAlaGluSerGlySerIleThrLeuGlnValGlnLeu                              859095                                                                         AlaThrProGlyAspLeuSerCysLeuTrpValPheLysHisSerSer                                100105110                                                                     LeuGlyCysGlnProHisPheAspLeuGlnAsnArgGlyIleValSer                               115120125                                                                      MetAlaIleLeuAsnValThrGl uThrGlnAlaGlyGluTyrLeuLeu                              130135140                                                                      HisIleGlnSerGluArgAlaAsnTyrThrValLeuPheThrValAsn                               145150155 160                                                                  ValArgAspThrGlnLeuTyrValLeuArgArgProTyrPheArgLys                               165170175                                                                      MetGluAsnGlnAspAlaLeuLeuCysIleSerGluGlyValP roGlu                              180185190                                                                      ProThrValGluTrpValLeuCysSerSerHisArgGluSerCysLys                               195200205                                                                      GluGluGlyPro AlaValValArgLysGluGluLysValLeuHisGlu                              210215220                                                                      LeuPheGlyThrAspIleArgCysCysAlaArgAsnAlaLeuGlyArg                               225230 235240                                                                  GluCysThrLysLeuPheThrIleAspLeuAsnGlnAlaProGlnSer                               245250255                                                                      ThrLeuProGlnLeuPheLeuLysValGlyGl uProLeuTrpIleArg                              260265270                                                                      CysLysAlaIleHisValAsnHisGlyPheGlyLeuThrTrpGluLeu                               275280285                                                                       GluAspLysAlaLeuGluGluGlySerTyrPheGluMetSerThrTyr                              290295300                                                                      SerThrAsnArgThrMetIleArgIleLeuLeuAlaPheValSerSer                               305 310315320                                                                  ValGlyArgAsnAspThrGlyTyrTyrThrCysSerSerSerLysHis                               325330335                                                                      ProSerGlnSerAlaLeuVal ThrIleLeuGluLysGlyPheIleAsn                              340345350                                                                      AlaThrSerSerGlnGluGluTyrGluIleAspProTyrGluLysPhe                               355360 365                                                                     CysPheSerValArgPheLysAlaTyrProArgIleArgCysThrTrp                               370375380                                                                      IlePheSerGlnAlaSerPheProCysGluGlnArgGlyLeuGluAsp                               385 390395400                                                                  GlyTyrSerIleSerLysPheCysAspHisLysAsnLysProGlyGlu                               405410415                                                                      TyrIlePhe TyrAlaGluAsnAspAspAlaGlnPheThrLysMetPhe                              420425430                                                                      ThrLeuAsnIleArgLysLysProGlnValLeuAlaAsnAlaSerAla                               435 440445                                                                     SerGlnAlaSerCysSerSerAspGlyTyrProLeuProSerTrpThr                               450455460                                                                      TrpLysLysCysSerAspLysSerProAsnCysThrGluGlu IlePro                              465470475480                                                                   GluGlyValTrpAsnLysLysAlaAsnArgLysValPheGlyGlnTrp                               485490495                                                                      ValSerSerSerThrLeuAsnMetSerGluAlaGlyLysGlyLeuLeu                               500505510                                                                      ValLysCysCysAlaTyrAsnSerMetGlyThrSerCysGluThrIle                               5 15520525                                                                     PheLeuAsnSerProGlyProPheProPheIleGlnAspAsnIleSer                               530535540                                                                      PheTyrAlaThrIleGlyLeuCysLeuPro PheIleValValLeuIle                              545550555560                                                                   ValLeuIleCysHisLysTyrLysLysGlnPheArgTyrGluSerGln                               565570 575                                                                     LeuGlnMetIleGlnValThrGlyProLeuAspAsnGluTyrPheTyr                               580585590                                                                      ValAspPheArgAspTyrGluTyrAspLeuLysTrpGluPhePro Arg                              595600605                                                                      GluAsnLeuGluPheGlyLysValLeuGlySerGlyAlaPheGlyArg                               610615620                                                                      ValMetAsnAlaThrAlaT yrGlyIleSerLysThrGlyValSerIle                              625630635640                                                                   GlnValAlaValLysMetLeuLysGluLysAlaAspSerCysGluLys                               645 650655                                                                     GluAlaLeuMetSerGluLeuLysMetMetThrHisLeuGlyHisHis                               660665670                                                                      AspAsnIleValAsnLeuLeuGlyAlaCysThr LeuSerGlyProVal                              675680685                                                                      TyrLeuIlePheGluTyrCysCysTyrGlyAspLeuLeuAsnTyrLeu                               690695700                                                                      ArgSerLy sArgGluLysPheHisArgThrTrpThrGluIlePheLys                              705710715720                                                                   GluHisAsnPheSerSerTyrProThrPheGlnAlaHisSerAsnSer                                725730735                                                                     SerMetProGlySerArgGluValGlnLeuHisProProLeuAspGln                               740745750                                                                      LeuSerGlyPheAsnGlyAsnS erIleHisSerGluAspGluIleGlu                              755760765                                                                      TyrGluAsnGlnLysArgLeuAlaGluGluGluGluGluAspLeuAsn                               7707757 80                                                                     ValLeuThrPheGluAspLeuLeuCysPheAlaTyrGlnValAlaLys                               785790795800                                                                   GlyMetGluPheLeuGluPheLysSerCysValHisArgAspLeuAla                                805810815                                                                     AlaArgAsnValLeuValThrHisGlyLysValValLysIleCysAsp                               820825830                                                                      PheGlyLeuAl aArgAspIleLeuSerAspSerSerTyrValValArg                              835840845                                                                      GlyAsnAlaArgLeuProValLysTrpMetAlaProGluSerLeuPhe                               850855 860                                                                     GluGlyIleTyrThrIleLysSerAspValTrpSerTyrGlyIleLeu                               865870875880                                                                   LeuTrpGluIlePheSerLeuGlyValAsnProTyrP roGlyIlePro                              885890895                                                                      ValAspAlaAsnPheTyrLysLeuIleGlnSerGlyPheLysMetGlu                               900905910                                                                       GlnProPheTyrAlaThrGluGlyIleTyrPheValMetGlnSerCys                              915920925                                                                      TrpAlaPheAspSerArgLysArgProSerPheProAsnLeuThrSer                               930 935940                                                                     PheLeuGlyCysGlnLeuAlaGluAlaGluGluAlaCysIleArgThr                               945950955960                                                                   SerIleHisLeuProLysGlnAlaAl aProGlnGlnArgGlyGlyLeu                              965970975                                                                      ArgAlaGlnSerProGlnArgGlnValLysIleHisArgGluArgSer                               980985 990                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 5406 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 208..4311                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: matpeptide                                                        (B) LOCATION: 208..4308                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        CTGTGTCCCGCAGCCGGATAACCTGGCTGACCCGATTCCGCGGACACCCGTGCAGCCGCG60                 GCTGGAGCCAGGGCGCCGGTGCCCGCGCTCTCCCCGGTCTTGCGCTGCGGGGGCCGATAC120                CGCCTCTGTGACTTC TTTGCGGGCCAGGGACGGAGAAGGAGTCTGTGCCTGAGAAACTGG180               GCTCTGTGCCCAGGCGCGAGGTGCAGGATGGAGAGCAAGGGCCTGCTAGCT231                         MetGluSerLysGlyLeuLeuAla                                                        15                                                                            GTCGCTCTGTGGTTCTGCGTGGAGACCCGAGCCGCCTCTGTGGGTTTG279                            ValAlaLeuTrpPheCysValGluThrArgAlaAlaSerValGlyLeu                               10 1520                                                                        CCTGGCGATTTTCTCCATCCCCCCAAGCTCAGCACACAGAAAGACATA327                            ProGlyAspPheLeuHisProProLysLeuSerThrGlnLysAspIle                               2530 3540                                                                      CTGACAATTTTGGCAAATACAACCCTTCAGATTACTTGCAGGGGACAG375                            LeuThrIleLeuAlaAsnThrThrLeuGlnIleThrCysArgGlyGln                               4 55055                                                                        CGGGACCTGGACTGGCTTTGGCCCAATGCTCAGCGTGATTCTGAGGAA423                            ArgAspLeuAspTrpLeuTrpProAsnAlaGlnArgAspSerGluGlu                               60 6570                                                                        AGGGTATTGGTGACTGAATGCGGCGGTGGTGACAGTATCTTCTGCAAA471                            ArgValLeuValThrGluCysGlyGlyGlyAspSerIlePheCysLys                               75 8085                                                                        ACACTCACCATTCCCAGGGTGGTTGGAAATGATACTGGAGCCTACAAG519                            ThrLeuThrIleProArgValValGlyAsnAspThrGlyAlaTyrLys                               9095 100                                                                       TGCTCGTACCGGGACGTCGACATAGCCTCCACTGTTTATGTCTATGTT567                            CysSerTyrArgAspValAspIleAlaSerThrValTyrValTyrVal                               105110 115120                                                                  CGAGATTACAGATCACCATTCATCGCCTCTGTCAGTGACCAGCATGGC615                            ArgAspTyrArgSerProPheIleAlaSerValSerAspGlnHisGly                               125 130135                                                                     ATCGTGTACATCACCGAGAACAAGAACAAAACTGTGGTGATCCCCTGC663                            IleValTyrIleThrGluAsnLysAsnLysThrValValIleProCys                               140 145150                                                                     CGAGGGTCGATTTCAAACCTCAATGTGTCTCTTTGCGCTAGGTATCCA711                            ArgGlySerIleSerAsnLeuAsnValSerLeuCysAlaArgTyrPro                               155160 165                                                                     GAAAAGAGATTTGTTCCGGATGGAAACAGAATTTCCTGGGACAGCGAG759                            GluLysArgPheValProAspGlyAsnArgIleSerTrpAspSerGlu                               170175 180                                                                     ATAGGCTTTACTCTCCCCAGTTACATGATCAGCTATGCCGGCATGGTC807                            IleGlyPheThrLeuProSerTyrMetIleSerTyrAlaGlyMetVal                               185190195 200                                                                  TTCTGTGAGGCAAAGATCAATGATGAAACCTATCAGTCTATCATGTAC855                            PheCysGluAlaLysIleAsnAspGluThrTyrGlnSerIleMetTyr                               205210 215                                                                     ATAGTTGTGGTTGTAGGATATAGGATTTATGATGTGATTCTGAGCCCC903                            IleValValValValGlyTyrArgIleTyrAspValIleLeuSerPro                               220225 230                                                                     CCGCATGAAATTGAGCTATCTGCCGGAGAAAAACTTGTCTTAAATTGT951                            ProHisGluIleGluLeuSerAlaGlyGluLysLeuValLeuAsnCys                               23524024 5                                                                     ACAGCGAGAACAGAGCTCAATGTGGGGCTTGATTTCACCTGGCACTCT999                            ThrAlaArgThrGluLeuAsnValGlyLeuAspPheThrTrpHisSer                               250255260                                                                      CCACCT TCAAAGTCTCATCATAAGAAGATTGTAAACCGGGATGTGAAA1047                          ProProSerLysSerHisHisLysLysIleValAsnArgAspValLys                               265270275280                                                                   CC CTTTCCTGGGACTGTGGCGAAGATGTTTTTGAGCACCTTGACAATA1095                          ProPheProGlyThrValAlaLysMetPheLeuSerThrLeuThrIle                               285290295                                                                      G AAAGTGTGACCAAGAGTGACCAAGGGGAATACACCTGTGTAGCGTCC1143                          GluSerValThrLysSerAspGlnGlyGluTyrThrCysValAlaSer                               300305310                                                                      AGT GGACGGATGATCAAGAGAAATAGAACATTTGTCCGAGTTCACACA1191                          SerGlyArgMetIleLysArgAsnArgThrPheValArgValHisThr                               315320325                                                                      AAGCCTTTT ATTGCTTTCGGTAGTGGGATGAAATCTTTGGTGGAAGCC1239                          LysProPheIleAlaPheGlySerGlyMetLysSerLeuValGluAla                               330335340                                                                      ACAGTGGGCAGTCAAGT CCGAATCCCTGTGAAGTATCTCAGTTACCCA1287                          ThrValGlySerGlnValArgIleProValLysTyrLeuSerTyrPro                               345350355360                                                                   GCTCCTGATATCA AATGGTACAGAAATGGAAGGCCCATTGAGTCCAAC1335                          AlaProAspIleLysTrpTyrArgAsnGlyArgProIleGluSerAsn                               365370375                                                                      TACACAATGATT GTTGGCGATGAACTCACCATCATGGAAGTGACTGAA1383                          TyrThrMetIleValGlyAspGluLeuThrIleMetGluValThrGlu                               380385390                                                                      AGAGATGCAGGAAAC TACACGGTCATCCTCACCAACCCCATTTCAATG1431                          ArgAspAlaGlyAsnTyrThrValIleLeuThrAsnProIleSerMet                               395400405                                                                      GAGAAACAGAGCCACATGGT CTCTCTGGTTGTGAATGTCCCACCCCAG1479                          GluLysGlnSerHisMetValSerLeuValValAsnValProProGln                               410415420                                                                      ATCGGTGAGAAAGCCTTGATCTCGCCTA TGGATTCCTACCAGTATGGG1527                          IleGlyGluLysAlaLeuIleSerProMetAspSerTyrGlnTyrGly                               425430435440                                                                   ACCATGCAGACATTGACATGCACA GTCTACGCCAACCCTCCCCTGCAC1575                          ThrMetGlnThrLeuThrCysThrValTyrAlaAsnProProLeuHis                               445450455                                                                      CACATCCAGTGGTACTGGCAGCTA GAAGAAGCCTGCTCCTACAGACCC1623                          HisIleGlnTrpTyrTrpGlnLeuGluGluAlaCysSerTyrArgPro                               460465470                                                                      GGCCAAACAAGCCCGTATGCTTGTAA AGAATGGAGACACGTGGAGGAT1671                          GlyGlnThrSerProTyrAlaCysLysGluTrpArgHisValGluAsp                               475480485                                                                      TTCCAGGGGGGAAACAAGATCGAAGTCACCA AAAACCAATATGCCCTG1719                          PheGlnGlyGlyAsnLysIleGluValThrLysAsnGlnTyrAlaLeu                               490495500                                                                      ATTGAAGGAAAAAACAAAACTGTAAGTACGCTGGTCATC CAAGCTGCC1767                          IleGluGlyLysAsnLysThrValSerThrLeuValIleGlnAlaAla                               505510515520                                                                   AACGTGTCAGCGTTGTACAAATGTGAAGCCATCAAC AAAGCGGGACGA1815                          AsnValSerAlaLeuTyrLysCysGluAlaIleAsnLysAlaGlyArg                               525530535                                                                      GGAGAGAGGGTCATCTCCTTCCATGTGATCAGGGG TCCTGAAATTACT1863                          GlyGluArgValIleSerPheHisValIleArgGlyProGluIleThr                               540545550                                                                      GTGCAACCTGCTGCCCAGCCAACTGAGCAGGAGAGTG TGTCCCTGTTG1911                          ValGlnProAlaAlaGlnProThrGluGlnGluSerValSerLeuLeu                               555560565                                                                      TGCACTGCAGACAGAAATACGTTTGAGAACCTCACGTGGTAC AAGCTT1959                          CysThrAlaAspArgAsnThrPheGluAsnLeuThrTrpTyrLysLeu                               570575580                                                                      GGCTCACAGGCAACATCGGTCCACATGGGCGAATCACTCACACCAGTT 2007                          GlySerGlnAlaThrSerValHisMetGlyGluSerLeuThrProVal                               585590595600                                                                   TGCAAGAACTTGGATGCTCTTTGGAAACTGAATGGCACCATGTTTTC T2055                          CysLysAsnLeuAspAlaLeuTrpLysLeuAsnGlyThrMetPheSer                               605610615                                                                      AACAGCACAAATGACATCTTGATTGTGGCATTTCAGAATGCCTCTC TG2103                          AsnSerThrAsnAspIleLeuIleValAlaPheGlnAsnAlaSerLeu                               620625630                                                                      CAGGACCAAGGCGACTATGTTTGCTCTGCTCAAGATAAGAAGACCAAG 2151                          GlnAspGlnGlyAspTyrValCysSerAlaGlnAspLysLysThrLys                               635640645                                                                      AAAAGACATTGCCTGGTCAAACAGCTCATCATCCTAGAGCGCATGGCA219 9                          LysArgHisCysLeuValLysGlnLeuIleIleLeuGluArgMetAla                               650655660                                                                      CCCATGATCACCGGAAATCTGGAGAATCAGACAACAACCATTGGCGAG2247                           ProMet IleThrGlyAsnLeuGluAsnGlnThrThrThrIleGlyGlu                              665670675680                                                                   ACCATTGAAGTGACTTGCCCAGCATCTGGAAATCCTACCCCACACATT2295                           Thr IleGluValThrCysProAlaSerGlyAsnProThrProHisIle                              685690695                                                                      ACATGGTTCAAAGACAACGAGACCCTGGTAGAAGATTCAGGCATTGTA2343                           Th rTrpPheLysAspAsnGluThrLeuValGluAspSerGlyIleVal                              700705710                                                                      CTGAGAGATGGGAACCGGAACCTGACTATCCGCAGGGTGAGGAAGGAG2391                           LeuA rgAspGlyAsnArgAsnLeuThrIleArgArgValArgLysGlu                              715720725                                                                      GATGGAGGCCTCTACACCTGCCAGGCCTGCAATGTCCTTGGCTGTGCA2439                           AspGlyGly LeuTyrThrCysGlnAlaCysAsnValLeuGlyCysAla                              730735740                                                                      AGAGCGGAGACGCTCTTCATAATAGAAGGTGCCCAGGAAAAGACCAAC2487                           ArgAlaGluThrLeuPhe IleIleGluGlyAlaGlnGluLysThrAsn                              745750755760                                                                   TTGGAAGTCATTATCCTCGTCGGCACTGCAGTGATTGCCATGTTCTTC2535                           LeuGluValIleIl eLeuValGlyThrAlaValIleAlaMetPhePhe                              765770775                                                                      TGGCTCCTTCTTGTCATTCTCGTACGGACCGTTAAGCGGGCCAATGAA2583                           TrpLeuLeuLeuV alIleLeuValArgThrValLysArgAlaAsnGlu                              780785790                                                                      GGGGAACTGAAGACAGGCTACTTGTCTATTGTCATGGATCCAGATGAA2631                           GlyGluLeuLysThr GlyTyrLeuSerIleValMetAspProAspGlu                              795800805                                                                      TTGCCCTTGGATGAGCGCTGTGAACGCTTGCCTTATGATGCCAGCAAG2679                           LeuProLeuAspGluArgCys GluArgLeuProTyrAspAlaSerLys                              810815820                                                                      TGGGAATTCCCCAGGGACCGGCTGAAACTAGGAAAACCTCTTGGCCGC2727                           TrpGluPheProArgAspArgLeuLysLe uGlyLysProLeuGlyArg                              825830835840                                                                   GGTGCCTTCGGCCAAGTGATTGAGGCAGACGCTTTTGGAATTGACAAG2775                           GlyAlaPheGlyGlnValIleGluA laAspAlaPheGlyIleAspLys                              845850855                                                                      ACAGCGACTTGCAAAACAGTAGCCGTCAAGATGTTGAAAGAAGGAGCA2823                           ThrAlaThrCysLysThrValAla ValLysMetLeuLysGluGlyAla                              860865870                                                                      ACACACAGCGAGCATCGAGCCCTCATGTCTGAACTCAAGATCCTCATC2871                           ThrHisSerGluHisArgAlaLeuMet SerGluLeuLysIleLeuIle                              875880885                                                                      CACATTGGTCACCATCTCAATGTGGTGAACCTCCTAGGCGCCTGCACC2919                           HisIleGlyHisHisLeuAsnValValAsnLe uLeuGlyAlaCysThr                              890895900                                                                      AAGCCGGGAGGGCCTCTCATGGTGATTGTGGAATTCTCGAAGTTTGGA2967                           LysProGlyGlyProLeuMetValIleValGluPheSerL ysPheGly                              905910915920                                                                   AACCTATCAACTTACTTACGGGGCAAGAGAAATGAATTTGTTCCCTAT3015                           AsnLeuSerThrTyrLeuArgGlyLysArgAsnGlu PheValProTyr                              925930935                                                                      AAGAGCAAAGGGGCACGCTTCCGCCAGGGCAAGGACTACGTTGGGGAG3063                           LysSerLysGlyAlaArgPheArgGlnGlyLysAsp TyrValGlyGlu                              940945950                                                                      CTCTCCGTGGATCTGAAAAGACGCTTGGACAGCATCACCAGCAGCCAG3111                           LeuSerValAspLeuLysArgArgLeuAspSerIleTh rSerSerGln                              955960965                                                                      AGCTCTGCCAGCTCAGGCTTTGTTGAGGAGAAATCGCTCAGTGATGTA3159                           SerSerAlaSerSerGlyPheValGluGluLysSerLeuSerA spVal                              970975980                                                                      GAGGAAGAAGAAGCTTCTGAAGAACTGTACAAGGACTTCCTGACCTTG3207                           GluGluGluGluAlaSerGluGluLeuTyrLysAspPheLeuThrLeu                               985 9909951000                                                                 GAGCATCTCATCTGTTACAGCTTCCAAGTGGCTAAGGGCATGGAGTTC3255                           GluHisLeuIleCysTyrSerPheGlnValAlaLysGlyMetGluPh e                              100510101015                                                                   TTGGCATCAAGGAAGTGTATCCACAGGGACCTGGCAGCACGAAACATT3303                           LeuAlaSerArgLysCysIleHisArgAspLeuAlaAlaArgAsn Ile                              102010251030                                                                   CTCCTATCGGAGAAGAATGTGGTTAAGATCTGTGACTTCGGCTTGGCC3351                           LeuLeuSerGluLysAsnValValLysIleCysAspPheGlyLeuAl a                              103510401045                                                                   CGGGACATTTATAAAGACCCGGATTATGTCAGAAAAGGAGATGCCCGA3399                           ArgAspIleTyrLysAspProAspTyrValArgLysGlyAspAlaArg                                105010551060                                                                  CTCCCTTTGAAGTGGATGGCCCCGGAAACCATTTTTGACAGAGTATAC3447                           LeuProLeuLysTrpMetAlaProGluThrIlePheAspArgValTyr                               1065 107010751080                                                              ACAATTCAGAGCGATGTGTGGTCTTTCGGTGTGTTGCTCTGGGAAATA3495                           ThrIleGlnSerAspValTrpSerPheGlyValLeuLeuTrpGluIle                                108510901095                                                                  TTTTCCTTAGGTGCCTCCCCATACCCTGGGGTCAAGATTGATGAAGAA3543                           PheSerLeuGlyAlaSerProTyrProGlyValLysIleAspGluGlu                                110011051110                                                                  TTTTGTAGGAGATTGAAAGAAGGAACTAGAATGCGGGCTCCTGACTAC3591                           PheCysArgArgLeuLysGluGlyThrArgMetArgAlaProAspTyr                                111511201125                                                                  ACTACCCCAGAAATGTACCAGACCATGCTGGACTGCTGGCATGAGGAC3639                           ThrThrProGluMetTyrGlnThrMetLeuAspCysTrpHisGluAsp                               1130 11351140                                                                  CCCAACCAGAGACCCTCGTTTTCAGAGTTGGTGGAGCATTTGGGAAAC3687                           ProAsnGlnArgProSerPheSerGluLeuValGluHisLeuGlyAsn                               1145115 011551160                                                              CTCCTGCAAGCAAATGCGCAGCAGGATGGCAAAGACTATATTGTTCTT3735                           LeuLeuGlnAlaAsnAlaGlnGlnAspGlyLysAspTyrIleValLeu                               1 16511701175                                                                  CCAATGTCAGAGACACTGAGCATGGAAGAGGATTCTGGACTCTCCCTG3783                           ProMetSerGluThrLeuSerMetGluGluAspSerGlyLeuSerLeu                               118 011851190                                                                  CCTACCTCACCTGTTTCCTGTATGGAGGAAGAGGAAGTGTGCGACCCC3831                           ProThrSerProValSerCysMetGluGluGluGluValCysAspPro                               1195 12001205                                                                  AAATTCCATTATGACAACACAGCAGGAATCAGTCATTATCTCCAGAAC3879                           LysPheHisTyrAspAsnThrAlaGlyIleSerHisTyrLeuGlnAsn                               1210 12151220                                                                  AGTAAGCGAAAGAGCCGGCCAGTGAGTGTAAAAACATTTGAAGATATC3927                           SerLysArgLysSerArgProValSerValLysThrPheGluAspIle                               12251230 12351240                                                              CCATTGGAGGAACCAGAAGTAAAAGTGATCCCAGATGACAGCCAGACA3975                           ProLeuGluGluProGluValLysValIleProAspAspSerGlnThr                               1245 12501255                                                                  GACAGTGGGATGGTCCTTGCATCAGAAGAGCTGAAAACTCTGGAAGAC4023                           AspSerGlyMetValLeuAlaSerGluGluLeuLysThrLeuGluAsp                               1260 12651270                                                                  AGGAACAAATTATCTCCATCTTTTGGTGGAATGATGCCCAGTAAAAGC4071                           ArgAsnLysLeuSerProSerPheGlyGlyMetMetProSerLysSer                               1275 12801285                                                                  AGGGAGTCTGTGGCCTCGGAAGGCTCCAACCAGACCAGTGGCTACCAG4119                           ArgGluSerValAlaSerGluGlySerAsnGlnThrSerGlyTyrGln                               12901295 1300                                                                  TCTGGGTATCACTCAGATGACACAGACACCACCGTGTACTCCAGCGAC4167                           SerGlyTyrHisSerAspAspThrAspThrThrValTyrSerSerAsp                               13051310131 51320                                                              GAGGCAGGACTTTTAAAGATGGTGGATGCTGCAGTTCACGCTGACTCA4215                           GluAlaGlyLeuLeuLysMetValAspAlaAlaValHisAlaAspSer                               13251 3301335                                                                  GGGACCACACTGCAGCTCACCTCCTGTTTAAATGGAAGTGGTCCTGTC4263                           GlyThrThrLeuGlnLeuThrSerCysLeuAsnGlySerGlyProVal                               1340134 51350                                                                  CCGGCTCCGCCCCCAACTCCTGGAAATCACGAGAGAGGTGCTGCTTAGATTTT4318                      ProAlaProProProThrProGlyAsnHisGluArgGlyAlaAla                                  13551360 1365                                                                  AGTGTTGTTCTTTCCACCACCCGGAAGTAGCCACATTTGATTTTCATTTTTGGAGGAGGG4378               ACCTCAGACTGCAAGGAGCTTGTCCTCAGGGCATTTCCAGAGAAGATGCCCATGACCCAA4438               GAATGTGTTGACTCTACTCTCTTTTCCATTCAT TTAAAAGTCCTATATAATGTGCCCTGC4498              TGTGGTCTCACTACCAGTTAAAGCAAAAGACTTTCAAACACGTGGACTCTGTCCTCCAAG4558               AAGTGGCAACGGCACCTCTGTGAAACTGGATCGAATGGGCAATGCTTTGTGTGTTGAGGA4618               TGGGTGAGAT GTCCCAGGGCCGAGTCTGTCTACCTTGGAGGCTTTGTGGAGGATGCGGCT4678              ATGAGCCAAGTGTTAAGTGTGGGATGTGGACTGGGAGGAAGGAAGGCGCAAGCCGTCCGG4738               AGAGCGGTTGGAGCCTGCAGATGCATTGTGCTGGCTCTGGTGGAGGTGGGCTTGTG GCCT4798              GTCAGGAAACGCAAAGGCGGCCGGCAGGGTTTGGTTTTGGAAGGTTTGCGTGCTCTTCAC4858               AGTCGGGTTACAGGCGAGTTCCCTGTGGCGTTTCCTACTCCTAATGAGAGTTCCTTCCGG4918               ACTCTTACGTGTCTCCTGGCCTGGCCCCAGGAA GGAAATGATGCAGCTTGCTCCTTCCTC4978              ATCTCTCAGGCTGTGCCTTAATTCAGAACACCAAAAGAGAGGAACGTCGGCAGAGGCTCC5038               TGACGGGGCCGAAGAATTGTGAGAACAGAACAGAAACTCAGGGTTTCTGCTGGGTGGAGA5098               CCCACGTGGC GCCCTGGTGGCAGGTCTGAGGGTTCTCTGTCAAGTGGCGGTAAAGGCTCA5158              GGCTGGTGTTCTTCCTCTATCTCCACTCCTGTCAGGCCCCCAAGTCCTCAGTATTTTAGC5218               TTTGTGGCTTCCTGATGGCAGAAAAATCTTAATTGGTTGGTTTGCTCTCCAGATAA TCAC5278              TAGCCAGATTTCGAAATTACTTTTTAGCCGAGGTTATGATAACATCTACTGTATCCTTTA5338               GAATTTTAACCTATAAAACTATGTCTACTGGTTTCTGCCTGTGTGCTTATGTTAAAAAAA5398               AAAAAAAA 5406                                                                  (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1367 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetGluSerLysGlyLeuLeuAlaValAlaLeuTrpPhe CysValGlu                              151015                                                                         ThrArgAlaAlaSerValGlyLeuProGlyAspPheLeuHisProPro                               202530                                                                         Ly sLeuSerThrGlnLysAspIleLeuThrIleLeuAlaAsnThrThr                              354045                                                                         LeuGlnIleThrCysArgGlyGlnArgAspLeuAspTrpLeuTrpPro                               50 5560                                                                        AsnAlaGlnArgAspSerGluGluArgValLeuValThrGluCysGly                               65707580                                                                       GlyGlyAspSerIlePheCysLysThrL euThrIleProArgValVal                              859095                                                                         GlyAsnAspThrGlyAlaTyrLysCysSerTyrArgAspValAspIle                               100105 110                                                                     AlaSerThrValTyrValTyrValArgAspTyrArgSerProPheIle                               115120125                                                                      AlaSerValSerAspGlnHisGlyIleValTyrIleThrGluAsnLys                                130135140                                                                     AsnLysThrValValIleProCysArgGlySerIleSerAsnLeuAsn                               145150155160                                                                   ValSerLeuCysAlaAr gTyrProGluLysArgPheValProAspGly                              165170175                                                                      AsnArgIleSerTrpAspSerGluIleGlyPheThrLeuProSerTyr                               180 185190                                                                     MetIleSerTyrAlaGlyMetValPheCysGluAlaLysIleAsnAsp                               195200205                                                                      GluThrTyrGlnSerIleMetTyrIleValValValValG lyTyrArg                              210215220                                                                      IleTyrAspValIleLeuSerProProHisGluIleGluLeuSerAla                               225230235240                                                                   GlyGlu LysLeuValLeuAsnCysThrAlaArgThrGluLeuAsnVal                              245250255                                                                      GlyLeuAspPheThrTrpHisSerProProSerLysSerHisHisLys                               260 265270                                                                     LysIleValAsnArgAspValLysProPheProGlyThrValAlaLys                               275280285                                                                      MetPheLeuSerThrLeuThrIleGluSe rValThrLysSerAspGln                              290295300                                                                      GlyGluTyrThrCysValAlaSerSerGlyArgMetIleLysArgAsn                               305310315 320                                                                  ArgThrPheValArgValHisThrLysProPheIleAlaPheGlySer                               325330335                                                                      GlyMetLysSerLeuValGluAlaThrValGlySerGlnValArgIle                                340345350                                                                     ProValLysTyrLeuSerTyrProAlaProAspIleLysTrpTyrArg                               355360365                                                                      AsnGlyArgProIleGlu SerAsnTyrThrMetIleValGlyAspGlu                              370375380                                                                      LeuThrIleMetGluValThrGluArgAspAlaGlyAsnTyrThrVal                               385390395 400                                                                  IleLeuThrAsnProIleSerMetGluLysGlnSerHisMetValSer                               405410415                                                                      LeuValValAsnValProProGlnIleGlyGluLysAl aLeuIleSer                              420425430                                                                      ProMetAspSerTyrGlnTyrGlyThrMetGlnThrLeuThrCysThr                               435440445                                                                      ValTyr AlaAsnProProLeuHisHisIleGlnTrpTyrTrpGlnLeu                              450455460                                                                      GluGluAlaCysSerTyrArgProGlyGlnThrSerProTyrAlaCys                               465470 475480                                                                  LysGluTrpArgHisValGluAspPheGlnGlyGlyAsnLysIleGlu                               485490495                                                                      ValThrLysAsnGlnTyrAlaLeuIle GluGlyLysAsnLysThrVal                              500505510                                                                      SerThrLeuValIleGlnAlaAlaAsnValSerAlaLeuTyrLysCys                               515520 525                                                                     GluAlaIleAsnLysAlaGlyArgGlyGluArgValIleSerPheHis                               530535540                                                                      ValIleArgGlyProGluIleThrValGlnProAlaAlaGlnProThr                               545 550555560                                                                  GluGlnGluSerValSerLeuLeuCysThrAlaAspArgAsnThrPhe                               565570575                                                                      GluAsnLeuThrTrp TyrLysLeuGlySerGlnAlaThrSerValHis                              580585590                                                                      MetGlyGluSerLeuThrProValCysLysAsnLeuAspAlaLeuTrp                               595600 605                                                                     LysLeuAsnGlyThrMetPheSerAsnSerThrAsnAspIleLeuIle                               610615620                                                                      ValAlaPheGlnAsnAlaSerLeuGlnAspGlnGlyAspTyrValCys                               625630635640                                                                   SerAlaGlnAspLysLysThrLysLysArgHisCysLeuValLysGln                               645650655                                                                      LeuI leIleLeuGluArgMetAlaProMetIleThrGlyAsnLeuGlu                              660665670                                                                      AsnGlnThrThrThrIleGlyGluThrIleGluValThrCysProAla                               675 680685                                                                     SerGlyAsnProThrProHisIleThrTrpPheLysAspAsnGluThr                               690695700                                                                      LeuValGluAspSerGlyIleValLeuArgAspGly AsnArgAsnLeu                              705710715720                                                                   ThrIleArgArgValArgLysGluAspGlyGlyLeuTyrThrCysGln                               725730 735                                                                     AlaCysAsnValLeuGlyCysAlaArgAlaGluThrLeuPheIleIle                               740745750                                                                      GluGlyAlaGlnGluLysThrAsnLeuGluValIleIleLeuValGly                                755760765                                                                     ThrAlaValIleAlaMetPhePheTrpLeuLeuLeuValIleLeuVal                               770775780                                                                      ArgThrValLysArgAlaAsnGluG lyGluLeuLysThrGlyTyrLeu                              785790795800                                                                   SerIleValMetAspProAspGluLeuProLeuAspGluArgCysGlu                               8058 10815                                                                     ArgLeuProTyrAspAlaSerLysTrpGluPheProArgAspArgLeu                               820825830                                                                      LysLeuGlyLysProLeuGlyArgGlyAlaPheGlyGln ValIleGlu                              835840845                                                                      AlaAspAlaPheGlyIleAspLysThrAlaThrCysLysThrValAla                               850855860                                                                      ValLysMetLeuLy sGluGlyAlaThrHisSerGluHisArgAlaLeu                              865870875880                                                                   MetSerGluLeuLysIleLeuIleHisIleGlyHisHisLeuAsnVal                               885 890895                                                                     ValAsnLeuLeuGlyAlaCysThrLysProGlyGlyProLeuMetVal                               900905910                                                                      IleValGluPheSerLysPheGlyAsnL euSerThrTyrLeuArgGly                              915920925                                                                      LysArgAsnGluPheValProTyrLysSerLysGlyAlaArgPheArg                               930935940                                                                      Gln GlyLysAspTyrValGlyGluLeuSerValAspLeuLysArgArg                              945950955960                                                                   LeuAspSerIleThrSerSerGlnSerSerAlaSerSerGlyPheVal                                965970975                                                                     GluGluLysSerLeuSerAspValGluGluGluGluAlaSerGluGlu                               980985990                                                                      LeuTyrLysAspPheLe uThrLeuGluHisLeuIleCysTyrSerPhe                              99510001005                                                                    GlnValAlaLysGlyMetGluPheLeuAlaSerArgLysCysIleHis                               10101015 1020                                                                  ArgAspLeuAlaAlaArgAsnIleLeuLeuSerGluLysAsnValVal                               1025103010351040                                                               LysIleCysAspPheGlyLeuAlaArgAspIleTyrLysAs pProAsp                              104510501055                                                                   TyrValArgLysGlyAspAlaArgLeuProLeuLysTrpMetAlaPro                               106010651070                                                                   Gl uThrIlePheAspArgValTyrThrIleGlnSerAspValTrpSer                              107510801085                                                                   PheGlyValLeuLeuTrpGluIlePheSerLeuGlyAlaSerProTyr                               1090 10951100                                                                  ProGlyValLysIleAspGluGluPheCysArgArgLeuLysGluGly                               1105111011151120                                                               ThrArgMetArgAlaProAspTyrTh rThrProGluMetTyrGlnThr                              112511301135                                                                   MetLeuAspCysTrpHisGluAspProAsnGlnArgProSerPheSer                               11401145 1150                                                                  GluLeuValGluHisLeuGlyAsnLeuLeuGlnAlaAsnAlaGlnGln                               115511601165                                                                   AspGlyLysAspTyrIleValLeuProMetSerGluThrLeuSerMe t                              117011751180                                                                   GluGluAspSerGlyLeuSerLeuProThrSerProValSerCysMet                               1185119011951200                                                               GluGluGluGl uValCysAspProLysPheHisTyrAspAsnThrAla                              120512101215                                                                   GlyIleSerHisTyrLeuGlnAsnSerLysArgLysSerArgProVal                               1220 12251230                                                                  SerValLysThrPheGluAspIleProLeuGluGluProGluValLys                               123512401245                                                                   ValIleProAspAspSerGlnThrAspSerGl yMetValLeuAlaSer                              125012551260                                                                   GluGluLeuLysThrLeuGluAspArgAsnLysLeuSerProSerPhe                               1265127012751 280                                                              GlyGlyMetMetProSerLysSerArgGluSerValAlaSerGluGly                               128512901295                                                                   SerAsnGlnThrSerGlyTyrGlnSerGlyTyrHisSerAspAspThr                                130013051310                                                                  AspThrThrValTyrSerSerAspGluAlaGlyLeuLeuLysMetVal                               131513201325                                                                   AspAlaAlaValHisAl aAspSerGlyThrThrLeuGlnLeuThrSer                              133013351340                                                                   CysLeuAsnGlySerGlyProValProAlaProProProThrProGly                               134513501 3551360                                                              AsnHisGluArgGlyAlaAla                                                          1365                                                                       

What I claim is:
 1. A recombinant nucleic acid molecule that is flk-1 having the sequence shown in FIG. 2 (SEQIDNO:3).
 2. An isolated mRNA that encodes the flk-1 protein, said protein having the amino acid sequence shown in FIG. 2 (SEQIDNO:3).
 3. A recombinant nucleic acid molecule comprising the sequence shown in FIG. 2 (SEQIDNO:3) from nucleotide 1 to nucleotide
 2493. 